EP3701037A1 - Verfahren zur verrringerung von milchsäure in einem biokraftstofffermentationssystem - Google Patents

Verfahren zur verrringerung von milchsäure in einem biokraftstofffermentationssystem

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Publication number
EP3701037A1
EP3701037A1 EP18797458.9A EP18797458A EP3701037A1 EP 3701037 A1 EP3701037 A1 EP 3701037A1 EP 18797458 A EP18797458 A EP 18797458A EP 3701037 A1 EP3701037 A1 EP 3701037A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
seq
variant
sequence identity
lpmo
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18797458.9A
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English (en)
French (fr)
Inventor
Armindo Ribeiro GASPAR
VictorGabriel Guadalupe MEDINA
Xin Li
Kelly Cristina LEITE MULDER
Angela SHOWS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Original Assignee
Novozymes AS
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Filing date
Publication date
Application filed by Novozymes AS filed Critical Novozymes AS
Publication of EP3701037A1 publication Critical patent/EP3701037A1/de
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/99—Miscellaneous (1.14.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

Definitions

  • the present invention relates to a process for reducing and/or preventing an increase in lactic acid devels (e.g., due to bacterial contamination) in a fermentation product production process, such as especially ethanol production, wherein a lytic polysaccharide monooxygenase is added before or during saccharification and/or fermentation to reduce and/or prevent an increase in lactic acid levels during fermentationn.
  • a fermentation product production process such as especially ethanol production
  • Fermentation products such as ethanol are typically produced by first grinding starch-containing material in a dry-grind or wet-milling process, then degrading the material into fermentable sugars using enzymes and finally converting the sugars directly or indirectly into the desired fermentation product using a fermenting organism.
  • Liquid fermentation products are recovered from the fermented mash (often referred to as "beer mash"), e.g., by distillation, which separates the desired fermentation product, e.g. ethanol, from other liquids and/or solids.
  • the remaining fraction is referred to as "whole stillage”.
  • Whole stillage typically contains about 10 to 20% solids. The whole stillage is separated into a solid and a liquid fraction, e.g., by centrifugation.
  • wet cake (or “wet grains") and the separated liquid fraction is referred to as "thin stillage”.
  • Wet cake and thin stillage contain about 35 and 7% solids, respectively.
  • Wet cake, with optional additional dewatering is used as a component in animal feed or is dried to provide "Distillers Dried Grains” (DDG) used as a component in animal feed.
  • DDG Dispensillers Dried Grains
  • Thin stillage is typically evaporated to provide evaporator condensate and syrup or may alternatively be recycled to the slurry tank as "backset”. Evaporator condensate may either be forwarded to a methanator before being discharged and/or may be recycled to the slurry tank as "cook water”.
  • the syrup may be blended into DDG or added to the wet cake before or during the drying process, which can comprise one or more dryers in sequence, to produce DDGS (Distillers Dried Grain with Solubles).
  • Syrup typically contains about 25 to 35% solids. Oil can also be extracted from the thin stillage and/or syrup as a by-product for use in biodiesel production, as a feed or food additive or product, or other biorenewable products.
  • Contaminating bacteria and their metabolic end-products such as lactic acid and/or acetic acid, lead to reduced fermentation yields which lead to considerable economic loss to the producer (see Thomas et al., 2001 , J. Aplied Microbiology, 90: 819-828).
  • the contaminating bacteria compete with the fermenting organism (e.g., yeast) for sugars in the fermentation medium.
  • the lactic acid and/or acetic acid produced by the unwanted bacteria also negatively impact yeast growth. Therefore, it is desirable to decrease the levels of lactic acid and/or unwanted bacteria that compete with fermenting organisms for sugar.
  • the present invention provides a solution to the problem of unwanted bacteria competing for sugar in the fermentation medium and the lactic acid they produce, by providing proving a biological solution to reduce and/or eliminate the lactic acid (e.g., due tobacterial cells)for instance, by adding at least one lytic polysaccharide monoxyenase (LPMO) polypeptide or an enzyme composition comprising a LPMO before or during saccharification and/or fermentation.
  • LPMO lytic polysaccharide monoxyenase
  • the present invention relates to a process for reducing and/or preventing an increase in lactic acid levels in a biofuel fermentation system, the process comprising introducing a lytic polysaccharide monooxygenase (LPMO) polypeptide or an enzyme composition comprising a LPMO polypeptide to a biofuel fermentation system, wherein the fermentation system comprises one or more fermentation vessels, pipes and/or components.
  • LPMO polypeptide or enzyme composition comprising the LPMO polypeptide is added at a concentration sufficient to reduce and/or prevent an increase in lactic acid levels in the biofuel fermentation system.
  • At least one of the fermentation vessels is a fermentation tank and the LPMO polypeptide or the enzyme composition comprising the LPMO polypeptide is introduced into the propagation or fermentation tank.
  • at least one of the fermentation vessels is a yeast propagation tank and the LPMO polypeptide or the enzyme composition comprising the LPMO polypeptide is introduced into the yeast propagation tank.
  • the biofuel is ethanol.
  • the present invention relates to a process for producing a fermentation product from a starch-containing material, the process comprising: a) liquefying a starch- containing material in the presence of an alpha-amylase to form a liquefied mash; b) saccharifying the liquefied mash using a carbohydrate source generating enzyme to produce a fermentable sugar; c) fermenting the sugar using a fermenting organism under conditions suitable to produce the fermentation product, wherein at least one LPMO polypeptide or enzyme composition comprising an LPMO polypeptide is added before or during saccharifying step b) and/or fermenting step c).
  • steps b) and c) are carried out simultaneously.
  • a slurry of the starch containing material is heated to above the gelatinization temperature.
  • the at least one LPMO polypeptide or enzyme composition comprising the LPMO polypeptide is added after liquefaction.
  • the at least one LPMO polypeptide or the enzyme composition comprising the LPMO polypeptide is added before or during saccharification.
  • the at least one LPMO polypeptide or the enzyme composition comprising the LPMO polypeptide is added before or during fermentation.
  • the fermenting organism is yeast and the at least one LPMO polypeptide or the enzyme composition comprising the LPMO polypeptide is added before or during yeast propagation.
  • the LPMO polypeptide or the enzyme composition comprising the LPMO polypeptide is introduced just after liquefaction and before the fermentation tank or propagation tank. In an embodiment, the LPMO polypeptide or the enzyme composition comprising the LPMO polypeptide is introduced at any point of the mash cooling system. In an embodiment, the LPMO polypeptide or the enzyme composition comprising the LPMO polypeptide is added to a heat exchanger. In an embodiment, the LPMO polypeptide or the enzyme composition comprising the LPMO polypeptide is added to a mixing tank.
  • the fermentation product is an alcohol, preferably ethanol.
  • the bacterial cells are gram-positive bacteria or gram-negative bacteria cells. In an embodiment, the bacterial cells are Lactobacillus cells, or cells that produce lactic acid.
  • the present invention relates to the use of an LPMO polypeptide or enzyme composition comprising an LPMO polypeptide for reducing the levels of lactic acid during fermentation in an ethanol production process.
  • the present invention relates to the use of an LPMO polypeptide or enzyme composition comprising an LPMO polypeptide for reducing the levels of lactic acid during yeast propagation.
  • the LPMO is selected from the group consisting of a Auxiliary Activity 9 (AA9) polypeptide, a Auxiliary Activity 10 (AA10) polypeptide, a Auxiliary Activity 11 (AA1 1) polypeptide, a Auxiliary Activity 13 (AA13) polypeptide, and combinations thereof.
  • the LPMO polypeptide is a AA9 polypeptide selected from the group consisting of: i) the Thermoascus aurantiacus AA9 polypeptide of SEQ ID NO: 1 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; ii) the Penicillium emersonii AA9 polypeptide of SEQ ID NO: 2 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; iii) the Thielavia terrestris AA9 polypeptide of SEQ ID NO: 3 or a variant thereof
  • the LPMO polypeptide is a AA13 polypeptide selected from the group consisting of: i) the Aspergillus terreus AA13 polypeptide of SEQ ID NO: 1 19 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; ii) the Aspergillus lentulus AA13 polypeptide of SEQ ID NO: 120 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; iii) the Aspergillus nidulans polypeptide of SEQ ID NO: 123 or a variant thereof having at least 60%, at least 65%
  • FIG. 1 shows an exemplary dry-grind ethanol production process.
  • FIG. 2 shows lactic acid concentrations after fermentation of corn mash in the presence of various AA9 polypeptides compared to control (0, 0).
  • FIG. 3 shows ethanol concentrations after fermentation of corn mash in the presence of various AA9 polypeptides compared to control (0, 0).
  • FIG. 4 shows lactic acid concentrations after fermentation of corn mash in the presence of various AA13 polypeptides compared to controls.
  • FIG. 5 shows lactic acid concentrations after fermentation of corn mash in the presence of increasing doses of an At-AA13 polypeptide compared to controls.
  • the present invention relates to reducing and/or eliminating bacterial contamination, for instance, in biofuel fermentation systems.
  • the present invention also relates to processes for producing a fermentation product from a starch-containing material using a fermenting organism, wherein at least one lytic polysaccharide monoxyenases (LPMO) is added before and/or during fermentation.
  • LPMO lytic polysaccharide monoxyenases
  • Auxiliary Activity 9 AA9
  • LPMOs according to the present disclosure perform equal to or better than antibiotics, such as penicillin, at reducing levels of lactic acid during fermentation.
  • the invention relates to a process for reducing and/or eliminating bacterial contamination in a biofuel fermentation system, the process comprising introducing a lytic polysaccharide monooxygenase (LPMO) polypeptide or an enzyme composition comprising a lytic polysaccharide monooxygenase (LPMO) polypeptide to a biofuel fermentation system.
  • LPMO lytic polysaccharide monooxygenase
  • the LPMO polypeptide can be added at a concentration sufficient to inhibit growth of contaminating bacterial cells in the biofuel fermentation system.
  • the present disclosure contemplates reducing and/or eliminating bacterial contamination due to the presence of variety of types contaminating bacterial cells present in biofuel fermentation systems.
  • the bacterial cells are gram-positive bacteria or gram-negative bacteria cells.
  • the contaminating bacterial cells are, but are not limited to, lactic acid and/or acetic acid producing bacteria of the genus Lactobacillus, which are known to contaminate fermentation systems.
  • Lactobacillus species that have been found to contaminate fermentation systems include strains of Lactobacillus collinoides, Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, and/or Lactobacillus rhamnosus, and mixtures thereof.
  • the phrase "reducing and/or eliminating bacterial contamination” encompasses the reduction of existing populations of bacterial cells present in the fermentation system, as well as inhibition of bacterial growth.
  • the LPMO polypeptide or the enzyme composition comprising at least one LPMO polypeptide can reduce the number of bacterial cells present in a fermentation system or inhibit bacterial growth by at least 1 %, 3%, 5%, 10%, 1 1 %, 13%, 15%, 17%, 21 %, 24%, 26%, 32%, 35%, 40%, 45%, 50%, 54%, 58%, 61 %, 63%, 66%, 70%, 75%, 77%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the fermentation system may include one or more fermentation vessels, pipes, and/or components, which are configured to perform a fermentation product production process, such as the exemplary dry-grind ethanol production process shown in FIG. 1 .
  • a fermentation product production process such as the exemplary dry-grind ethanol production process shown in FIG. 1 .
  • the LPMO polypeptide or enzyme composition comprising the LPMO polypeptide may be introduced into, or prior to, the propagation or fermentation system at a variety of different locations.
  • at least one of the fermentation vessels in the fermentation system is a fermentation tank and the enzyme composition is introduced into the fermentation tank.
  • the LPMO polypeptide or enzyme composition comprising the LPMO polypeptide is introduced to the fermentation tank before fermentation begins.
  • At least one of the fermentation vessels is a yeast propagation tank and the LPMO polypeptide or enzyme composition comprising the LPMO polypeptide is introduced into the yeast propagation tank.
  • the LPMO polypeptide or enzyme composition comprising the LPMO polypeptide is introduced just after liquefaction and before the fermentation tank or propagation tank.
  • the LPMO polypeptide or enzyme composition comprising the LPMO polypeptide is introduced at any point of the mash cooling system.
  • the LPMO polypeptide or enzyme composition comprising the LPMO polypeptide is added to a heat exchanger.
  • the LPMO polypeptide or enzyme composition comprising the LPMO polypeptide is added to a mixing tank.
  • the biofuel is an alcohol.
  • the alcohol is ethanol.
  • the alcohol is methanol.
  • the alcohol is butanol.
  • any LPMO polypeptide for instance the LPMO polypeptides described in Section III below, can be used in a composition or process described in this section.
  • the invention relates to a process for reducing and/or preventing an increase, in lactic acid in a biofuel fermentation system, the process comprising introducing a LPMO polypeptide or an enzyme composition comprising a lytic polysaccharide monooxygenase (LPMO) polypeptide to a biofuel fermentation system.
  • LPMO polypeptide or enzyme composition comprising the LPMO polypeptide can be added at a concentration sufficient to reduce and/or prevent an increase in lactic acid in the biofuel fermentation system.
  • an LPMO polypeptide or enzyme composition comprising at least one LPMO can reduce the level of lactic acid in a fermentation system by at least 1 %, 3%, 5%, 10%, 11 %, 13%, 15%, 17%, 21 %, 24%, 26%, 32%, 35%, 40%, 45%, 50%, 54%, 58%, 61 %, 63%, 66%, 70%, 75%, 77%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • an LPMO polypeptide or enzyme composition comprising at least one LPMO can prevent the increase in the level of lactic acid in a fermentation system by at least 1 %, 3%, 5%, 10%, 11 %, 13%, 15%, 17%, 21 %, 24%, 26%, 32%, 35%, 40%, 45%, 50%, 54%, 58%, 61 %, 63%, 66%, 70%, 75%, 77%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% or more.
  • the fermentation system may include one or more fermentation vessels, pipes, and/or components, which are configured to perform a fermentation product production process, such as the exemplary dry-grind ethanol production process shown in FIG. 1.
  • a fermentation product production process such as the exemplary dry-grind ethanol production process shown in FIG. 1.
  • the LPMO polypeptide or enzyme composition comprising thea LPMO polypeptide may be introduced into the fermentation system at a variety of different locations.
  • at least one of the fermentation vessels in the fermentation system is a fermentation tank and the LPMO polypeptide or enzyme composition comprising the LPMO polypeptide is introduced into the fermentation tank.
  • the LPMO polypeptide or enzyme composition comprising the LPMO polypeptide is introduced to the fermentation tank before fermentation begins.
  • At least one of the fermentation vessels is a yeast propagation tank and the LPMO polypeptide or enzyme composition comprising the LPMO polypeptide is introduced into the yeast propagation tank.
  • the LPMO polypeptide or enzyme composition comprising the LPMO polypeptide is introduced just after liquefaction and before the fermentation tank or propagation tank.
  • the LPMO polypeptide or enzyme composition comprising the LPMO polypeptide is introduced at any point of the mash cooling system.
  • the biofuel is an alcohol.
  • the alcohol is ethanol.
  • the alcohol is methanol.
  • the alcohol is butanol.
  • any LPMO polypeptide for instance the LPMO polypeptides described in Section III below, can be used in a composition or process described in this section.
  • lytic polysaccharide monoxygenases LPMO
  • lytic polysaccharide monooxygenase or "LPMO” is used synonymously herein with “lytic polyscahharide monooxygenase polypeptide” and "LPMO polypeptide”, which refer to an enzyme that oxidizes sp(3) carbons in polysaccharides such as chitin, cellulose, and starch in the presence of an external electron donor and is believed to utilize copper at the active site to activate molecular oxygen.
  • Exemplary LPMOs belong to Auxiliary Activity families AA9, AA10, AA1 1 , and AA13, as defined in the database of carbohydrate active enzymes (http://www.cazy.org/).
  • the LPMO is selected from the group consisting of Auxiliary Activity 9 (AA9), Auxiliary Activity 10 (AA10), Auxiliary Activity 1 1 (AA1 1), Auxiliary Activity 13 (AA13), and combinations thereof.
  • the LPMO polypeptide is a AA9 polypeptide.
  • AA9 polypeptide means a polypeptide classified as a lytic polysaccharide monooxygenase (Quinlan et al., 2011 , Proc. Natl. Acad. Sci. USA 08: 15079- 15084; Phillips et al., 201 1 , ACS Chem. Biol. 6: 1399-1406; Li et al., 2012, Structure 20: 1051-1061).
  • AA9 polypeptides were formerly classified into the glycoside hydrolase Family 61 (GH61) according to Henrissat, 1991 , Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996, Biochem. J. 316: 695- 696.
  • Any AA9 polypeptide can be used as a component of the enzyme composition or used in the processes of the present invention, e.g., bacterial, fungal, archaea, etc.
  • AA9 lytic polysaccharide monooxygenases useful in the processes of the present invention include, but are not limited to, AA9 lytic polysaccharide monooxygenases from Thielavia terrestris (WO 2005/074647, WO 2008/148131 , and WO 2011/035027), Thermoascus aurantiacus (WO 2005/074656 and WO 2010/065830), Trichoderma reesei (WO 2007/089290 and WO 2012/149344), Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO 2009/085864, WO 2009/085868, and WO 2009/033071), Aspergillus fumigatus (WO 2010/138754), Penicillium pinophilum (WO 2011/005867), Thermoascus sp.
  • Thielavia terrestris WO 2005/074647, WO 2008/148131
  • the AA9 polypeptide is from the genus Thermoascus, such as Thermoascus aurantiacus or Thermoascus crustaceus, for example: the Thermoascus aurantiacus AA9 polypeptide of SEQ ID NO: 1 , SEQ I D NO: 13, SEQ ID NO: 20, or SEQ ID NO: 22, or variants thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto; or the Thermoa
  • the AA9 polypeptide is from the genus Penicillium, such as Penicillium emersonii, for example: the Penicillium emersonii AA9 polypeptide of SEQ ID NO: 3 or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto; the Penicillium pinophilum AA9 polypeptide of SEQ ID NO: 21 , or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 87
  • the AA9 polypeptide is from the genus Thielavia, such as Thielavia terrestris, for example, the Thielavia terrestris AA9 polypeptide of SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34, or variants thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 60%,
  • the AA9 polypeptide is from the genus Aspergillus, such as Aspergillus fumigatus, for example: the Aspergillus fumigatus AA9 polypeptide of SEQ ID NO: 5 or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto; or the Aspergillus aculeatus AA9 polypeptide of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID
  • the AA9 polypeptide is the Penicillium emersonii AA9 polypeptide of SEQ ID NO: 6 expressed in a Trichoderma reesei background, or a variant of SEQ ID NO: 6 having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA9 polypeptide is from the genus Trichoderma, such as Trichoderma reesei, for example: the Trichoderma reesei AA9 polypeptide of SEQ ID NO: 14 or SEQ ID NO: 84, or variants thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • Trichoderma reesei for example: the Trichoderma reesei AA9 polypeptide of SEQ ID NO: 14 or SEQ ID NO: 84, or
  • the AA9 polypeptide is from the genus Myceliophthora, such as
  • Myceliophthora thermophila for example: the Myceliophthora thermophila AA9 polypeptide of SEQ ID: 15, SEQ ID: 16, SEQ ID: 17, SEQ ID: 18, SEQ ID: 19, SEQ ID: 88, SEQ ID: 93, or SEQ ID: 94, or variants thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA9 polypeptide is from the genus Aurantiporus, such as Aurantiporus alborubescens, for example, the Aurantiporus alborubescens AA9 polypeptide of SEQ ID: 45, or SEQ ID: 46, or variants thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA9 polypeptide is from the genus Trichophaea, such as
  • Trichophaea saccata for example, the Trichophaea saccata AA9 polypeptide of SEQ ID: 47, or SEQ ID: 48, or variants thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA9 polypeptide is from the genus Talaromyces, such as Talaromyces stipitatus, for example: the Talaromyces stipitatus AA9 polypeptide of SEQ ID: 50, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto; the Talaromyces leycettanus AA9 polypeptide of SEQ ID NO: 82, or a variant thereof having at least 60%, e.g., at least 65%,
  • the AA9 polypeptide is from the genus Thermomyces, such as Thermomyces lanuginosus, for example, the Thermomyces lanuginosus AA9 polypeptide of SEQ ID: 52, or SEQ ID: 53, or variants thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA9 polypeptide is from the genus Humicola, such as Humicola insolens, for example, the Humicola insolens AA9 polypeptide of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 , SEQ ID NO: 72, SEQ ID NO:73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO:77, SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80
  • the AA9 polypeptide is from the genus Malbranchea, such as Malbranchea cinnamomea, for example, the Malbranchea cinnamomea AA9 polypeptide of SEQ ID: 81 , or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • Malbranchea cinnamomea for example, the Malbranchea cinnamomea AA9 polypeptide of SEQ ID: 81 , or a variant thereof having at least 60%, e.g
  • the AA9 polypeptide is from the genus Chaetomium, such as Chaetomium thermophilum, for example, the Chaetomium thermophilum AA9 polypeptide of SEQ ID: 83, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • Chaetomium thermophilum for example, the Chaetomium thermophilum AA9 polypeptide of SEQ ID: 83, or a variant thereof having at least 60%, e.g., at least
  • the AA9 polypeptide is from the genus Acrophialophora, such as Acrophialophora fusispora, for example, the Acrophialophora fusispora AA9 polypeptide of SEQ ID: 85, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA9 polypeptide is from the genus Corynascus, such as Corynascus sepedonium, for example, the Corynascus sepedonium AA9 polypeptide of SEQ ID: 86, or SEQ ID: 87, or variants thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA9 polypeptide is a AA9 variant comprising a substitution at one or more positions corresponding to positions 23, 61 , 62, 63, 64, 103, 104, 105, 106, 108, 109, 156, 185, 186, and 194 of the full-length polypeptide of SEQ ID NO: 4 herein, wherein the variant reduces and/or eliminates contaminating bacterial cells in a fermentation medium.
  • the AA9 variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the mature polypeptide of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56,
  • mature polypeptide means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C- terminal truncation, glycosylation, phosphorylation, etc.
  • the mature polypeptide is amino acids 22 to 250 of SEQ ID NO: 4 based on the SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 4 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 326 of SEQ ID NO: 7 based on the SignalP 3.0 program (Bendtsen et ai, 2004, J. Mol. Biol.
  • the mature polypeptide is amino acids 18 to 239 of SEQ ID NO: 8 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 8 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 258 of SEQ ID NO: 9 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 9 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 226 of SEQ ID NO: 10 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 10 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 304 of SEQ ID NO: 11 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 1 1 are a signal peptide.
  • the mature polypeptide is amino acids 16 to 317 of SEQ ID NO: 12 based on the SignalP program that predicts amino acids 1 to 15 of SEQ ID NO: 12 are a signal peptide.
  • the mature polypeptide is amino acids 22 to 249 of SEQ ID NO: 13 based on the SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 13 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 249 of SEQ ID NO: 14 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 14 are a signal peptide.
  • the mature polypeptide is amino acids 18 to 232 of SEQ ID NO: 15 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 15 are a signal peptide.
  • the mature polypeptide is amino acids 16 to 235 of SEQ ID NO: 16 based on the SignalP program that predicts amino acids 1 to 15 of SEQ ID NO: 16 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 323 of SEQ ID NO: 17 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 17 are a signal peptide.
  • the mature polypeptide is amino acids 16 to 310 of SEQ ID NO: 18 based on the SignalP program that predicts amino acids 1 to 15 of SEQ ID NO: 18 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 246 of SEQ ID NO: 19 based on the SignalP program that predicts amino acids 1 to 19 of SEQ I D NO: 19 are a signal peptide.
  • the mature polypeptide is amino acids 22 to 354 of SEQ ID NO: 20 based on the SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 20 are a signal peptide.
  • the mature polypeptide is amino acids 22 to 322 of SEQ ID NO: 21 based on the SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 21 are a signal peptide.
  • the mature polypeptide is amino acids 24 to 444 of SEQ ID NO: 22 based on the SignalP program that predicts amino acids 1 to 23 of SEQ ID NO: 22 are a signal peptide.
  • the mature polypeptide is amino acids 26 to 253 of SEQ ID NO: 23 based on the SignalP program that predicts amino acids 1 to 25 of SEQ ID NO: 23 are a signal peptide.
  • the mature polypeptide is amino acids 18 to 246 of SEQ ID NO: 24 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 24 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 334 of SEQ ID NO: 25 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 25 are a signal peptide.
  • the mature polypeptide is amino acids 18 to 227 of SEQ I D NO: 26 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 26 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 223 of SEQ ID NO: 27 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 27 are a signal peptide.
  • the mature polypeptide is amino acids 22 to 368 of SEQ ID NO: 28 based on the SignalP program that predicts amino acids 1 to 21 of SEQ I D NO: 28 are a signal peptide.
  • the mature polypeptide is amino acids 25 to 330 of SEQ ID NO: 29 based on the SignalP program that predicts amino acids 1 to 24 of SEQ ID NO: 29 are a signal peptide.
  • the mature polypeptide is amino acids 17 to 236 of SEQ ID NO: 30 based on the SignalP program that predicts amino acids 1 to 16 of SEQ ID NO: 30 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 250 of SEQ ID NO: 31 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 31 are a signal peptide.
  • the mature polypeptide is amino acids 23 to 478 of SEQ ID NO: 32 based on the SignalP program that predicts amino acids 1 to 22 of SEQ ID NO: 32 are a signal peptide.
  • the mature polypeptide is amino acids 17 to 230 of SEQ ID NO: 33 based on the SignalP program that predicts amino acids 1 to 16 of SEQ ID NO: 33 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 257 of SEQ ID NO: 34 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 34 are a signal peptide.
  • the mature polypeptide is amino acids 23 to 251 of SEQ ID NO: 35 based on the SignalP program that predicts amino acids 1 to 22 of SEQ ID NO: 35 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 349 of SEQ ID NO: 36 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 36 are a signal peptide.
  • the mature polypeptide is amino acids 24 to 436 of SEQ ID NO: 37 based on the SignalP program that predicts amino acids 1 to 23 of SEQ ID NO: 37 are a signal peptide.
  • the mature polypeptide is amino acids 21 to 344 of SEQ ID NO: 38 based on the SignalP program that predicts amino acids 1 to 23 of SEQ ID NO: 38 are a signal peptide.
  • the mature polypeptide is amino acids 26 to 400 of SEQ ID NO: 39 based on the SignalP program that predicts amino acids 1 to 25 of SEQ ID NO: 39 are a signal peptide.
  • the mature polypeptide is amino acids 21 to 389 of SEQ ID NO: 40 based on the SignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 40 are a signal peptide.
  • the mature polypeptide is amino acids 22 to 406 of SEQ ID NO: 41 based on the SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 41 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 427 of SEQ ID NO: 42 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 42 are a signal peptide.
  • the mature polypeptide is amino acids 18 to 267 of SEQ ID NO: 43 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 43 are a signal peptide.
  • the mature polypeptide is amino acids 21 to 273 of SEQ ID NO: 44 based on the SignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 44 are a signal peptide.
  • the mature polypeptide is amino acids 21 to 322 of SEQ ID NO: 45 based on the SignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 45 are a signal peptide.
  • the mature polypeptide is amino acids 18 to 234 of SEQ I D NO: 46 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 46 are a signal peptide.
  • the mature polypeptide is amino acids 24 to 233 of SEQ ID NO: 47 based on the SignalP program that predicts amino acids 1 to 23 of SEQ ID NO: 47 are a signal peptide.
  • the mature polypeptide is amino acids 17 to 237 of SEQ ID NO: 48 based on the SignalP program that predicts amino acids 1 to 16 of SEQ ID NO: 48 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 484 of SEQ ID NO: 49 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 49 are a signal peptide.
  • the mature polypeptide is amino acids 22 to 320 of SEQ ID NO: 50 based on the SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 50 are a signal peptide.
  • the mature polypeptide is amino acids 23 to 272 of SEQ ID NO: 51 based on the SignalP program that predicts amino acids 1 to 22 of SEQ ID NO: 51 are a signal peptide.
  • the mature polypeptide is amino acids 22 to 327 of SEQ ID NO: 52 based on the SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 52 are a signal peptide.
  • the mature polypeptide is amino acids 23 to 274 of SEQ ID NO: 53 based on the SignalP program that predicts amino acids 1 to 22 of SEQ ID NO: 53 are a signal peptide.
  • the mature polypeptide is amino acids 18 to 227 of SEQ ID NO: 54 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 54 are a signal peptide.
  • the mature polypeptide is amino acids 17 to 257 of SEQ ID NO: 55 based on the SignalP program that predicts amino acids 1 to 16 of SEQ ID NO: 55 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 246 of SEQ I D NO: 56 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 56 are a signal peptide.
  • the mature polypeptide is amino acids 28 to 265 of SEQ ID NO: 57 based on the SignalP program that predicts amino acids 1 to 27 of SEQ ID NO: 57 are a signal peptide.
  • the mature polypeptide is amino acids 16 to 310 of SEQ ID NO: 58 based on the SignalP program that predicts amino acids 1 to 15 of SEQ ID NO: 58 are a signal peptide.
  • the mature polypeptide is amino acids 21 to 354 of SEQ ID NO: 59 based on the SignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 59 are a signal peptide.
  • the mature polypeptide is amino acids 22 to 267 of SEQ ID NO: 60 based on the SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 60 are a signal peptide.
  • the mature polypeptide is amino acids 16 to 237 of SEQ ID NO: 61 based on the SignalP program that predicts amino acids 1 to 15 of SEQ ID NO: 61 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 234 of SEQ ID NO: 62 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 62 are a signal peptide.
  • the mature polypeptide is amino acids 18 to 226 of SEQ ID NO: 63 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 63 are a signal peptide.
  • the mature polypeptide is amino acids 17 to 231 of SEQ ID NO: 64 based on the SignalP program that predicts amino acids 1 to 16 of SEQ ID NO: 64 are a signal peptide.
  • the mature polypeptide is amino acids 22 to 248 of SEQ ID NO: 65 based on the SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 65 are a signal peptide.
  • the mature polypeptide is amino acids 18 to 233 of SEQ I D NO: 66 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 66 are a signal peptide.
  • the mature polypeptide is amino acids 21 to 243 of SEQ ID NO: 67 based on the SignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 67 are a signal peptide.
  • the mature polypeptide is amino acids 21 to 363 of SEQ ID NO: 68 based on the SignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 68 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 296 of SEQ ID NO: 69 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 69 are a signal peptide.
  • the mature polypeptide is amino acids 16 to 318 of SEQ ID NO: 70 based on the SignalP program that predicts amino acids 1 to 15 of SEQ ID NO: 70 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 259 of SEQ ID NO: 71 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 71 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 325 of SEQ ID NO: 72 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 72 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 298 of SEQ ID NO: 74 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 74 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 298 of SEQ ID NO: 74 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 74 are a signal peptide.
  • the mature polypeptide is amino acids 22 to 344 of SEQ ID NO: 75 based on the SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 75 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 330 of SEQ I D NO: 76 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 76 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 216 of SEQ ID NO: 77 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 77 are a signal peptide.
  • the mature polypeptide is amino acids 18 to 490 of SEQ ID NO: 78 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 78 are a signal peptide.
  • the mature polypeptide is amino acids 21 to 306 of SEQ ID NO: 79 based on the SignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 79 are a signal peptide.
  • the mature polypeptide is amino acids 22 to 339 of SEQ ID NO: 80 based on the SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 80 are a signal peptide.
  • the mature polypeptide is amino acids 23 to 334 of SEQ ID NO: 81 based on the SignalP program that predicts amino acids 1 to 22 of SEQ ID NO: 81 are a signal peptide.
  • the mature polypeptide is amino acids 24 to 366 of SEQ ID NO: 82 based on the SignalP program that predicts amino acids 1 to 23 of SEQ ID NO: 82 are a signal peptide.
  • the mature polypeptide is amino acids 21 to 364 of SEQ ID NO: 83 based on the SignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 83 are a signal peptide.
  • the mature polypeptide is amino acids 22 to 344 of SEQ ID NO: 84 based on the SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 84 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 252 of SEQ ID NO: 85 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 85 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 344 of SEQ I D NO: 86 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 86 are a signal peptide.
  • the mature polypeptide is amino acids 22 to 347 of SEQ ID NO: 87 based on the SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 87 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 342 of SEQ ID NO: 88 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 88 are a signal peptide.
  • the mature polypeptide is amino acids 27 to 254 of SEQ ID NO: 89 based on the SignalP program that predicts amino acids 1 to 26 of SEQ ID NO: 89 are a signal peptide.
  • the mature polypeptide is amino acids 23 to 272 of SEQ ID NO: 90 based on the SignalP program that predicts amino acids 1 to 22 of SEQ ID NO: 90 are a signal peptide.
  • the mature polypeptide is amino acids 23 to 272 of SEQ ID NO: 91 based on the SignalP program that predicts amino acids 1 to 22 of SEQ ID NO: 91 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 381 of SEQ ID NO: 128 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 128 are a signal peptide.
  • the mature polypeptide is amino acids amino acids 22 to 386 of SEQ ID NO: 130 based on the SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 130 are a signal peptide.
  • the mature polypeptide is amino acids 20 to 387 of SEQ ID NO: 131 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 131 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 253 of SEQ ID NO: 132 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 132 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 377 of SEQ ID NO: 133 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 133 are a signal peptide.
  • the mature polypeptide is amino acids 18 to 388 of SEQ ID NO: 134 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 134 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 391 of SEQ ID NO: 135 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 135 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 387 of SEQ ID NO: 136 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 136 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 390 of SEQ ID NO: 139 based on the SignalP program that predicts amino acids 1 to 18 of SEQ I D NO: 139 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 386 of SEQ ID NO: 141 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 141 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 394 of SEQ ID NO: 144 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 144 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 391 of SEQ ID NO: 145 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 145 are a signal peptide.
  • the mature polypeptide is amino acids 16 to 393 of SEQ ID NO: 148 based on the SignalP program that predicts amino acids 1 to 15 of SEQ ID NO: 148 are a signal peptide.
  • the mature polypeptide is amino acids 18 to 382 of SEQ ID NO: 149 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 149 are a signal peptide.
  • the mature polypeptide is amino acids 18 to 379 of SEQ ID NO: 150 based on the SignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 150 are a signal peptide.
  • the mature polypeptide is amino acids 19 to 383 of SEQ ID NO: 151 based on the SignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 151 are a signal peptide. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N- terminal amino acid) expressed by the same polynucleotide.
  • the AA9 polypeptide is the mature AA9 polypeptide of any one of SEQ ID NOs: 1-91 , or a variant of any of SEQ ID NOs: 1-91 having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the LPMO polypeptide is a AA10 polypeptide.
  • AA10 polypeptide means a polypeptide classified as a lytic polysaccharide monooxygenase (Quinlan et al., 2011 , Proc. Natl. Acad. Sci. USA 08: 15079-15084; Phillips et al., 201 1 , ACS Chem. Biol. 6: 1399-1406; Li et al., 2012, Structure 20: 1051-1061).
  • the AA10 may comprise a CBM33 domain comprising a carbohydrate-binding module (CBM), which is defined as a contiguous amino acid sequence within a carbohydrate binding protein with a discreet fold having carbohydrate-binding activity.
  • CBM carbohydrate-binding module
  • chitinases are known which contain one or more chitin binding modules in addition to catalytic regions.
  • ChiA of Serratia marcescens contains a fibronectin type Ill-type CBM
  • ChiB of Serratia marcescens contains a family 5 CBM
  • ChiC of Serratia marcescens contains a family 12 and a fibronectin type Ill-like CBM. See Bourne and Henrissat, 2001, Curr. Opin. Struct. Biol.
  • CBMs that bind to cellulose.
  • Proteins binding to chitin and containing CBMs that stimulate such binding may for example be structural or signaling molecules or they can be enzymes and the overall function of the protein may be determined by domains that are present in addition to the carbohydrate binding module.
  • AA10 polypeptide can be used as a component of the enzyme composition or used in the processes of the present invention, e.g., bacterial, fungal, archaea, etc.
  • AA10 polypeptides of use herein may be identified according to the CAZY classification system (cazy.org/CAZY/fam/acc_CBM.html), which is based on sequence similarities (Davies and Henrissat, 2002, Biochem Soc 730: 291-297 and Bourne and Henrissat, 2001 , supra). Proteins in this family are known to bind to chitin, but binding to other polysaccharides, including cellulose, has also been observed (Moser et al., 2008, Biotechnol. Bioeng.
  • AA10 polypeptides contain a family 33 carbohydrate binding module (CBM33).
  • CBM33 carbohydrate binding module
  • the CBM33 module makes up the whole protein, i.e., the protein consists of or consists essentially of a single family 33 CBM, which is in nature synthesized and secreted as such.
  • some family 33 CBMs may be fused to one or more additional non-catalytic carbohydrate binding modules (e.g., CBM family 2, CBM family 3 and CBM family 5 modules).
  • proteins are bi- or multi-domain proteins.
  • carbohydrate binding module that is present as an individual module within a much larger catalytic protein. This is the beta-1 ,4-mannanase protein of Caldibacillus cellulovorans (Sunna et al., 2000, Appl. Environ. Micro. 66(2): 664-670).
  • the family 33 CBMs are usually approximately 150-250 amino acids, e.g., 160-240,
  • the size of a protein can readily be determined by standard methods that are known in the art.
  • the AA10 polypeptide consists of a single family 33 CBM, or consists essentially of a family 33 CBM. If said AA10 polypeptide "consists essentially of a family 33 CBM, it is meant that additional amino acids may be present in the protein, in addition to those that make up the family 33 CBM. Preferably there are 1-3, 1-5, 1 -10, 10-20, 20-30, 30-40, 40-50,
  • additional amino acids are in general present C terminal to the family 33 CBM.
  • the AA10 polypeptide can comprise a family 33 CBM. Additional modules or domains may thus be present in the protein. Examples of such modules are CBM family 2,
  • CBM family 3 and CBM family 5 modules are in general found C-terminal to the family 33 CBM.
  • the AA10 polypeptide can contain, consist or consist essentially of a naturally occurring family 33 CBM (or CBM33 family protein) such as CBP21 (or to a homologue thereof from another species) or a biologically active fragment thereof. It can alternatively contain, consist or consist essentially of a variant of a naturally occurring family 33
  • CBM or CBM33 family protein
  • AA10 polypeptides which comprise or consist of a family 33 CBM module or the full family 33 CBM protein (which comprises the family 33 CBM module) or its fragments or variants are referred to herein, collectively, as CBM33 proteins or CBM33 family members or proteins.
  • Naturally occurring CBM33 proteins that can be used in the invention include microbial (e.g., bacterial), eukaryotic (e.g., Dictyostelium) or viral CBM33 proteins.
  • Bacterial CBM33 proteins are, however, preferred. Examples of known CBM33 proteins which may be used in the compositions and methods of the invention and relevant database accession numbers (which are hereby incorporated by reference) are set out in Table 1 of WO 2012/019151 (incorporated herein by reference in its entirety).
  • Bacterial CBM33 proteins can be from any appropriate source but are preferably from a genus selected from the group consisting of Bacillus, Chromobacterium, Enterococcus, Francisella, Hahella, Lactobacillus, Lactococcus, Legionella, Listeria, Oceanobacillus, Photobacterium, Photothabdus, Proteus, Pseudoalteromonas, Pseudomonas, Rickettsia, Saccharophagus, Salinvibrio, Serratia, Shewanella, Sodalis, Streptomyces, Thermobifida, Vibrio and Yersini and optionally Cellulomonas and Cellvibrio.
  • the CBM33 protein is a CBP21 as described in U.S. Patent Application No. 2007/0218046 which is incorporated herein by reference.
  • the CBP21 of Serratia marescens (SEQ ID NO: 4 in WO2012/019151) may be used.
  • EfCBM33 of Enterococcus faecalis (SEQ ID NO: 5 in WO2012/019151 ), E7 of Thermobifida fusca (SEQ ID NO: 6 in WO2012/019151 ), CelS2 of Streptomyces coelicolor A3(2) (SEQ ID NO: 7 in WO2012/019151 ), Cfla_0175 of Cellulomonas flavigena DSM 20109) (SEQ ID NO: 8 in WO2012/019151 ), Cfla_0172 of Cellulomonas flavigena DSM 20109) (SEQ ID NO: 9), Cfla_0316 of Cellulomonas flavigena DSM 20109) (SEQ ID NO: 10 in WO2012/019151), Cfla_0490 of Cellulomonas flavigena DSM 20109) (SEQ ID NO: 1 1 in WO2012/019151), CJA_2191 (Cbp33
  • ChbA of B. amyloliquefaciens Chu et al., 2001, Microbiology 147 (Pt 7):1793-803 CHB1 , 2 and 3 of Streptomyces (Svergun et al., 2000, Biochemistry 39(35): 10677-83, Zeltins et al., 1997, Eur. J. Biochem. 246(2):557-64, Zeltins et al., 1995, Anal. Biochem. 231 (2):287-94, Schnellmann et al., 1994, Mol. Microbiol.
  • the AA10 polypeptides can thus be or correspond to or comprise a naturally occurring
  • CBM33 family protein such as CBP21 , EfCBM33, ChbA, CHB1 , 2 and 3 and CBP1 or E7, CelS2, Cfla_0175, Cfla_0172, Cfla_0316, Cfla_0490, CJA_2191 (Cbp33A), CJA_3139 (Cbp33/10B) and SC01734) that it is found in nature or a biologically active fragment thereof.
  • the AA10 polypeptide may be a non-native variant.
  • the AA10 polypeptide is from the genus Streptomyces, such as
  • Streptomyces coelicolor or another Streptomyces sp., for example: the Streptomyces coelicolor AA 10 polypeptide of SEQ ID NO: 1 14, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto; or the Streptomyces sp.
  • AA10 polypeptide of SEQ ID NO: 115 or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA10 polypeptide is the polypeptide of SEQ ID NO: 1 16, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.
  • the AA10 polypeptide is the polypeptide of SEQ ID NO: 1 16, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but not100%, sequence identity thereto.
  • the AA10 polypeptide is the polypeptide of SEQ ID NO: 1 17, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.
  • the AA10 polypeptide is the polypeptide of SEQ ID NO: 117, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but not 100%, sequence identity thereto.
  • the LPMO polypeptide is a AA11 polypeptide.
  • AA11 polypeptide The term "Auxiliary Activity 11 polypeptide” or "AA1 1 polypeptide” means a polypeptide classified as a lytic polysaccharide monooxygenase (Quinlan et al., 2011 , Proc. Natl. Acad. Sci. USA 08: 15079-15084; Phillips et al., 201 1 , ACS Chem. Biol. 6: 1399-1406; Li et al., 2012, Structure 20: 1051-1061).
  • any AA1 1 polypeptide can be used as a component of the enzyme composition or used in the processes of the present invention, e.g., bacterial, fungal, archaea, etc.
  • the AA11 polypeptide is of fungal origin.
  • Exemplary AA11 polypeptides suitable for use in the compositions and processes herein are from the genera Aspergillus, such as, A. niger, A. nidulans, A. terreus, A. clavatus, A. oryzae or A. flavus, Neurospora, such as N. crassa or N.
  • tetrasperma Sclerotina, Gibberella, Coniothyrium, Psiticum, Magnaporthe, Podospora, Chaetomium, Phaeosphaeria, Botryotinia, Neosartorya, Pyrenophora, Panicum, Aureococcus, Penicillium, Trichoderma, Sordaria, Colleotrichum, Verticillium, Arthrobotrys, Nectria, Leptosphaeria, Fusarium, Glomerella, Geomyces, and Myceliophthora.
  • the AA11 polypeptide is from the genus Acremonium, such as Acremonium alcalophilum, for example the Acremonium alcalophilum AA10 polypeptide of SEQ ID NO: 1 18, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA11 polypeptide is the Acremonium alcalophilum AA10 polypeptide of SEQ ID NO: 1 18.
  • the LPMO polypeptide is a AA13 polypeptide.
  • AA13 polypeptide means a polypeptide classified as a lytic polysaccharide monooxygenase (Quinlan et al., 2011 , Proc. Natl. Acad. Sci. USA 08: 15079-15084; Phillips et al., 201 1 , ACS Chem. Biol. 6: 1399-1406; Li et al., 2012, Structure 20: 1051-1061).
  • any AA13 polypeptide can be used as a component of the enzyme composition or used in the processes of the present invention, e.g., bacterial, fungal, archaea, etc.
  • the AA13 polypeptide is of fungal origin.
  • Exemplary AA13 polypeptides suitable for use in the compositions and processes herein are from the genera Aspergillus, such as, A. niger, A. nidulans, A. terreus, A. clavatus, A. oryzae or A. flavus, Neurospora, such as N. crassa or N.
  • tetrasperma Sclerotina, Gibberella, Coniothyrium, Psiticum, Magnaporthe, Podospora, Chaetomium, Phaeosphaeria, Botryotinia, Neosartorya, Pyrenophora, Panicum, Aureococcus, Penicillium, Trichoderma, Sordaria, Colleotrichum, Verticillium, Arthrobotrys, Nectria, Leptosphaeria, Fusarium, Glomerella, Geomyces, and Myceliophthora.
  • the AA13 polypeptide is from the genus Aspergillus, such as Aspergillus terreus, Aspergillus lentulus, Aspergillus fischerianus, Aspergillus nidulans, Aspergillus insuetus, etc., for example: the Aspergillus terreus AA13 polypeptide of SEQ ID NO: 1 19, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto; the Aspergillus
  • the AA13 polypeptide is amino acids 22 to 386 of SEQ ID NO:
  • 130 or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA13 polypeptide is from the genus Penicillium, such as Penicillium polonicum, Penicillium oxalicum, Penicillium arizonense, etc., for example: the Penicillium polonicum AA13 polypeptide of SEQ ID NO: 124 or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto; the Penicillium oxalicum AA13 polypeptide of SEQ ID NO: 125, or a variant thereof
  • the AA13 polypeptide is amino acids 19 to 391 of SEQ ID NO:
  • the AA13 polypeptide is amino acids 19 to 387 of SEQ ID NO:
  • 136 or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
  • the AA13 polypeptide is amino acids 19 to 390 of SEQ ID NO:
  • 139 or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
  • the AA13 polypeptide is amino acids 19 to 386 of SEQ I D NO:
  • 141 or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least
  • the AA13 polypeptide is amino acids 19 to 394 of SEQ ID NO: 144, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA13 polypeptide is amino acids 19 to 391 of SEQ ID NO: 145, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA13 polypeptide is from the genus Mycothermus, such as
  • Mycothermus thermophilus for example, the Mycothermus thermophilus AA13 polypeptide of SEQ ID NO: 127, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA13 polypeptide is from the genus Acremonium, such as Acremonium sp. XZ1982, for example, the Acremonium sp. XZ7982AA13 AA13 polypeptide of SEQ ID NO: 128, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA13 polypeptide is amino acids 19 to 381 of SEQ ID NO: 128, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA13 polypeptide is from the genus Acrostalagmus, such as
  • Acrostalagmus luteoalbus for example, the Acrostalagmus luteoalbus AA13 polypeptide of SEQ ID NO: 129, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA13 polypeptide is from the genus Cladosporium, such as Cladosporium gossypiicola, for example, the Cladosporium gossypiicola AA13 polypeptide of SEQ ID NO: 131 , or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • Cladosporium gossypiicola for example, the Cladosporium gossypiicola AA13 polypeptide of SEQ ID
  • the AA13 polypeptide is amino acids 20 to 387 of SEQ ID NO:
  • 131 or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least
  • the AA13 polypeptide is from the genus Fusarium, such as Fusarium sp-75363, for example, the Fusarium sp-75363 AA13 polypeptide of SEQ ID NO:
  • 132 or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA13 polypeptide is amino acids 19 to 253 of SEQ ID NO: 132, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA13 polypeptide is from the genus Myrothecium, such as Myrothecium sp., for example, the Myrothecium sp AA13 polypeptide of SEQ ID NO: 133, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA13 polypeptide is amino acids 19 to 377 of SEQ ID NO:
  • 133 or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least
  • the AA13 polypeptide is from the genus Paraphoma, such as Paraphoma sp., for example, the Paraphoma sp. AA13 polypeptide of SEQ ID NO: 134, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA13 polypeptide is amino acids 18 to 388 of SEQ ID NO:
  • 134 or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA13 polypeptide is from the genus Pestalotiopsis, such as Pestalotiopsis sp-71627, for example, the Pestalotiopsis sp-71627 AA13 polypeptide of SEQ ID NO: 148, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • Pestalotiopsis sp-71627 for example, the Pestalotiopsis sp-71627 AA13 polypeptide of SEQ ID NO: 148
  • the AA13 polypeptide is amino acids 16 to 393 of SEQ ID NO: 148, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA13 polypeptide is from the genus Setophaeosphaeria, such as Setophaeosphaeria sp. NN051506, for example, the Setophaeosphaeria sp. ⁇ / ⁇ /057506 ⁇ 13 AA13 polypeptide of SEQ ID NO: 149, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • Setophaeosphaeria such as Setophaeosphaeria sp. NN05150
  • the AA13 polypeptide is amino acids 18 to 382 of SEQ ID NO:
  • the AA13 polypeptide is from the genus Talaromyces, such as Talaromyces sayulitensis, for example, the Talaromyces sayulitensis AA13 polypeptide of SEQ ID NO: 150, or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA13 polypeptide is amino acids 18 to 379 of SEQ ID NO:
  • 150 or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • the AA13 polypeptide is from the genus Trichocladium, such as Trichocladium asperum, for example, the Trichocladium asperum AA13 polypeptide of SEQ ID NO: 151 , or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity thereto.
  • Trichocladium asperum for example, the Trichocladium asperum AA13 polypeptide of SEQ ID NO: 151 , or a variant thereof having at least 60%, e.g., at least
  • the AA13 polypeptide is amino acids 19 to 383 of SEQ ID NO:
  • 151 or a variant thereof having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least
  • the present invention also relates to compositions comprising at least one lytic polysaccharide monooxygenase (LPMO) polypeptide of the present invention.
  • the compositions are enriched in the at least one LPMO polypeptide of the invention.
  • the term "enriched" indicates that the activity of the composition has been increased, e.g., with an enrichment factor of at least 1.1 , such as at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 2.0, at least 3.0, at least 4.0, at least 5.0, at least 10.
  • the composition comprises at least one, at least two, at least three, or at least four LPMO polypeptides of the invention.
  • compositions may further comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta- galactosidase, beta-glucanase, beta-glucosidase, glucan 1 ,4-a-glucosidase, glucan 1 ,4- alpha-maltohydrolase, glucan 1 ,4-a-glucosidase, glucan 1 ,4-alpha-maltohydrolase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase (mann
  • the composition comprises one or more formulating agents as disclosed herein, preferably one or more of the compounds selected from the list consisting of glycerol, ethylene glycol, 1 , 2-propylene glycol or 1 , 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolin and cellulose.
  • formulating agents as disclosed herein, preferably one or more of the compounds selected from the list consisting of glycerol, ethylene glycol, 1 , 2-propylene glycol or 1 , 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextri
  • the composition comprises one or more components selected from the list consisting of vitamins, minerals and amino acids.
  • the invention also relates to processes for producing a fermentation product from starch-containing material using a fermenting organism, wherein a lytic polysaccharide monooxygenase (LPMO) or an enzyme composition comprising at least one lytic polysaccharide monooxygenase (LPMO) is added before and/or during saccharification and/or fermentation.
  • a lytic polysaccharide monooxygenase LPMO
  • an enzyme composition comprising at least one lytic polysaccharide monooxygenase (LPMO) is added before and/or during saccharification and/or fermentation.
  • the invention relates to processes for producing fermentation products from starch-containing material without gelatinization (i.e., without cooking) of the starch- containing material (often referred to as a "raw starch hydrolysis" process), wherein at least one lytic polysaccharide monooxygenase is added.
  • the fermentation product such as ethanol, can be produced without liquefying the aqueous slurry containing the starch- containing material and water.
  • a process of the invention includes saccharifying (e.g.
  • milled starch-containing material e.g., granular starch
  • the desired fermentation product e.g., ethanol
  • un-gelatinized i.e., uncooked
  • milled cereal grains, such as corn.
  • the invention relates to processes for producing a fermentation product from starch-containing material comprising simultaneously saccharifying and fermenting starch-containing material using a carbohydrate-source generating enzymes and a fermenting organism at a temperature below the initial gelatinization temperature of said starch-containing material in the presence of a variant protease of the invention. Saccharification and fermentation may also be separate.
  • the invention relates to processes of producing fermentation products, comprising the following steps:
  • step (i) and/or (ii) is carried out using at least a glucoamylase and at least one LPMO polypeptide of the invention or an enzyme composition comprising at least one LPMO polypeptide of the invention.
  • said at least one LPMO polypeptide or enzyme composition comprising an LPMO polypeptide is added at a concentration sufficient to inhibit growth of contaminating bacterial cells.
  • said at least one LPMO or enzyme composition comprising at least one LPMO polypeptide is added at a concentration sufficient to reduce the levels of lactic acid during saccharification, fermentation, and/or simultaneous saccharification or fermentation (SSF).
  • SSF simultaneous saccharification or fermentation
  • the at least one LPMO polypeptide or enzyme composition comprising at least one LPMO polypeptide is added during saccharifying step (i). In an embodiment, the at least one LPMO polypeptide or enzyme composition comprising at least one LPMO polypeptide is added during fermenting step (ii).
  • an alpha amylase in particular a fungal alpha-amylase, is also added in step (i). Steps (i) and (ii) may be performed simultaneously.
  • the at least one LPMO is added during simultaneous saccharification and fermentation (SSF).
  • the process further includes propagating a fermenting organism under conditions suitable to be further used in fermentation.
  • the fermenting organism is yeast and the at least one LPMO enzyme composition comprising at least one LPMO polypeptide is added during yeast propagation.
  • said at least one LPMO or enzyme composition comprising at least one LPMO polypeptide is added at a concentration sufficient to reduce the levels of lactic acid during propagation.
  • the invention relates to processes for producing fermentation products, especially ethanol, from starch-containing material, which process includes a liquefaction step and sequentially or simultaneously performed saccharification and fermentation steps. Consequently, the invention relates to a process for producing a fermentation product from starch-containing material comprising the steps of:
  • At least one LPMO enzyme composition comprising at least one LPMO polypeptide is added before or during saccharifying step (b) and/or fermenting step (c).
  • said at least one LPMO or enzyme composition comprising at least one LPMO polypeptide is added at a concentration sufficient to inhibit growth of contaminating bacterial cells.
  • said at least one LPMO or enzyme composition comprising at least one LPMO polypeptide is added at a concentration sufficient to reduce the levels of lactic acid during saccharification, fermentation, and/or simultaneous saccharification or fermentation (SSF).
  • the at least one LPMO enzyme composition comprising at least one LPMO polypeptide is added before or during saccharifying step (b).
  • the at least one LPMO enzyme composition comprising at least one LPMO polypeptide is added before or during fermenting step (c).
  • an alpha amylase in particular a fungal alpha-amylase, is also added in step (b). Steps (b) and (c) may be performed simultaneously.
  • the at least one LPMO enzyme composition comprising at least one LPMO polypeptide is added during simultaneous saccharification and fermentation (SSF).
  • the process further includes propagating a fermenting organism under conditions suitable to be further used in fermentation.
  • the fermenting organism is yeast and the at least one LPMO enzyme composition comprising at least one LPMO polypeptide is added before or during yeast propagation.
  • said at least one LPMO or enzyme composition comprising at least one LPMO polypeptide is added at a concentration sufficient to reduce the levels of lactic acid during propagation.
  • the slurry is heated to above the gelatinization temperature and an alpha-amylase variant may be added to initiate liquefaction (thinning).
  • the slurry may in an embodiment be jet-cooked to further gelatinize the slurry before being subjected to alpha-amylase in step (a).
  • Liquefaction may in an embodiment be carried out as a three-step hot slurry process.
  • the slurry is heated to between 60-95°C, preferably between 70-90°C, such as preferably between 80-85°C at a pH of 4-6, in particular at a pH of 4.5-5.5, and alpha-amylase variant, optionally together with a hemicellulase, an endoglucanase, a protease, a carbohydrate- source generating enzyme, such as a glucoamylase, a phospholipase, a phytase, and/or pullulanase, are added to initiate liquefaction (thinning).
  • the liquefaction process is usually carried out at a pH of 4-6, in particular at a pH from 4.5 to 5.5.
  • Saccharification step (b) may be carried out using conditions well known in the art. For instance, a full saccharification process may last up to from about 24 to about 72 hours, however, it is common only to do a pre-saccharification of typically 40-90 minutes at a temperature between 30-65°C, typically about 60°C, followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation process (SSF process). Saccharification is typically carried out at a temperature from 20-75°C, in particular 40-70°C, typically around 60°C, and at a pH between 4 and 5, normally at about pH 4.5.
  • SSF process simultaneous saccharification and fermentation process
  • SSF simultaneous saccharification and fermentation
  • a fermenting organism such as yeast, and enzyme(s)
  • SSF may typically be carried out at a temperature from 25°C to 40°C, such as from 28°C to 35°C, such as from 30°C to 34°C, preferably around about 32°C.
  • fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours.
  • Any suitable starch-containing starting material may be used in a process of the present invention.
  • the starting material is generally selected based on the desired fermentation product.
  • starch-containing starting materials suitable for use in the processes of the present invention, include barley, beans, cassava, cereals, corn, milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca, wheat, and whole grains, or any mixture thereof.
  • the starch-containing material may also be a waxy or non-waxy type of corn and barley.
  • the starch-containing material is corn.
  • the starch-containing material is wheat.
  • Fermentation product means a product produced by a method or process including fermenting using a fermenting organism. Fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B12, beta-carotene); and hormones.
  • alcohols e.g., ethanol, methanol, butanol
  • organic acids e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid
  • ketones e.g., acetone
  • amino acids e.
  • the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry.
  • Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer.
  • the fermentation product is ethanol.
  • fermenting organism refers to any organism, including bacterial and fungal organisms, such as yeast and filamentous fungi, suitable for producing a desired fermentation product. Suitable fermenting organisms are able to ferment, i.e., convert, fermentable sugars, such as arabinose, fructose, glucose, maltose, mannose, or xylose, directly or indirectly into the desired fermentation product.
  • fermentable sugars such as arabinose, fructose, glucose, maltose, mannose, or xylose
  • fermenting organisms include fungal organisms such as yeast.
  • yeast include strains of Saccharomyces, in particular Saccharomyces cerevisiae or Saccharomyces uvarum; strains of Pichia, in particular Pichia stipitis such as Pichia stipitis CBS 5773 or Pichia pastoris; strains of Candida, in particular Candida arabinofermentans, Candida boidinii, Candida diddensii, Candida shehatae, Candida sonorensis, Candida tropicalis, or Candida utilis.
  • Other fermenting organisms include strains of Hansenula, in particular Hansenula anomala or Hansenula polymorphs, strains of Kluyveromyces, in particular Kluyveromyces fragilis or Kluyveromyces marxianus; and strains of Schizosaccharomyces, in particular Schizosaccharomyces pombe.
  • the fermenting organism is a C6 sugar fermenting organism, such as a strain of, e.g., Saccharomyces cerevisiae.
  • the fermenting organism is a C5 sugar fermenting organism, such as a strain of, e.g., Saccharomyces cerevisiae.
  • the fermentation conditions are determined based on, e.g., the kind of plant material, the available fermentable sugars, the fermenting organism(s) and/or the desired fermentation product.
  • One skilled in the art can easily determine suitable fermentation conditions.
  • the fermentation may be carried out at conventionally used conditions.
  • Preferred fermentation processes are anaerobic processes.
  • fermentations may be carried out at temperatures as high as 75°C, e.g., between 40-70°C, such as between 50-60°C.
  • temperatures as high as 75°C, e.g., between 40-70°C, such as between 50-60°C.
  • bacteria with a significantly lower temperature optimum down to around room temperature around 20°C are also known. Examples of suitable fermenting organisms can be found in the "Fermenting Organisms" section above.
  • the fermentation may go on for 24 to 96 hours, in particular for 35 to 60 hours.
  • the fermentation is carried out at a temperature between 20 to 40°C, preferably 26 to 34°C, in particular around 32°C.
  • the pH is from pH 3 to 6, preferably around pH 4 to 5.
  • the fermentation product may be separated from the fermentation medium.
  • the slurry may be distilled to extract the desired fermentation product (e.g., ethanol).
  • the desired fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques.
  • the fermentation product may also be recovered by stripping or other method well known in the art.
  • the fermentation product e.g., ethanol, with a purity of up to, e.g., about 96 vol. percent ethanol is obtained.
  • the method of the invention further comprises distillation to obtain the fermentation product, e.g., ethanol.
  • the fermentation and the distillation may be carried out simultaneously and/or separately/sequentially; optionally followed by one or more process steps for further refinement of the fermentation product.
  • the material remaining is considered the whole stillage.
  • whole stillage includes the material that remains at the end of the distillation process after recovery of the fermentation product, e.g., ethanol.
  • the fermentation product can optionally be recovered by any method known in the art.
  • the whole stillage is separated or partitioned into a solid and liquid phase by one or more methods for separating the thin stillage from the wet cake.
  • Separating whole stillage into thin stillage and wet cake in order to remove a significant portion of the liquid/water may be done using any suitable separation technique, including centrifugation, pressing and filtration.
  • the separation/dewatering is carried out by centrifugation.
  • Preferred centrifuges in industry are decanter type centrifuges, preferably high speed decanter type centrifuges.
  • An example of a suitable centrifuge is the NX 400 steep cone series from Alfa Laval which is a high-performance decanter.
  • the separation is carried out using other conventional separation equipment such as a plate/frame filter presses, belt filter presses, screw presses, gravity thickeners and deckers, or similar equipment.
  • Thin stillage is the term used for the supernatant of the centrifugation of the whole stillage.
  • the thin stillage contains 4-6 percent dry solids (DS) (mainly proteins, soluble fiber, fine fibers, and cell wall components) and has a temperature of about 60-90 degrees centigrade.
  • the thin stillage stream may be condensed by evaporation to provide two process streams including: (i) an evaporator condensate stream comprising condensed water removed from the thin stillage during evaporation, and (ii) a syrup stream, comprising a more concentrated stream of the non-volatile dissolved and non-dissolved solids, such as non-fermentable sugars and oil, remaining present from the thin stillage as the result of removing the evaporated water.
  • oil can be removed from the thin stillage or can be removed as an intermediate step to the evaporation process, which is typically carried out using a series of several evaporation stages.
  • Syrup and/or de-oiled syrup may be introduced into a dryer together with the wet grains (from the whole stillage separation step) to provide a product referred to as distillers dried grain with solubles, which also can be used as animal feed.
  • syrup and/or de-oiled syrup is sprayed into one or more dryers to combine the syrup and/or de-oiled syrup with the whole stillage to produce distillers dried grain with solubles.
  • the recycled thin stillage may constitute from about 1-70 vol.-%, preferably 15-60% vol.-%, especially from about 30 to 50 vol.-% of the slurry formed in step (a).
  • the process further comprises recycling at least a portion of the thin stillage stream treated with a LPMO of the invention to the slurry, optionally after oil has been extracted from the thin stillage stream.
  • the wet cake containing about 25-40 wt-%, preferably 30-38 wt-% dry solids, has been separated from the thin stillage (e.g., dewatered) it may be dried in a drum dryer, spray dryer, ring drier, fluid bed drier or the like in order to produce "Distillers Dried Grains" (DDG).
  • DDG is a valuable feed ingredient for animals, such as livestock, poultry and fish. It is preferred to provide DDG with a content of less than about 10-12 wt.-% moisture to avoid mold and microbial breakdown and increase the shelf life. Further, high moisture content also makes it more expensive to transport DDG.
  • the wet cake is preferably dried under conditions that do not denature proteins in the wet cake.
  • the wet cake may be blended with syrup separated from the thin stillage and dried into DDG with Solubles (DDGS).
  • DDG DDG with Solubles
  • Partially dried intermediate products such as are sometimes referred to as modified wet distillers grains, may be produced by partially drying wet cake, optionally with the addition of syrup before, during or after the drying process.
  • an alpha-amylase is present and/or added in liquefaction optionally together with a hemicellulase, an endoglucanase, a protease, a carbohydrate- source generating enzyme, such as a glucoamylase, a phospholipase, a phytase, and/or pullulanase.
  • the alpha-amylase added during liquefaction step i) may be any alpha-amylase.
  • Preferred are bacterial alpha-amylases, such as especially Bacillus alpha-amylases, such as Bacillus stearothermophilus alpha-amylases, which are stable at temperature used during liquefaction.
  • Bacterial Alpha-Amylases such as especially Bacillus alpha-amylases, such as Bacillus stearothermophilus alpha-amylases, which are stable at temperature used during liquefaction.
  • bacterial alpha-amylase means any bacterial alpha-amylase classified under EC 3.2.1.1.
  • a bacterial alpha-amylase used according to the invention may, e.g., be derived from a strain of the genus Bacillus, which is sometimes also referred to as the genus Geobacillus.
  • Bacillus alpha-amylase is derived from a strain of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus sp. TS-23, or Bacillus subtilis, but may also be derived from other Bacillus sp.
  • bacterial alpha-amylases include the Bacillus stearothermophilus alpha-amylase of SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 95 herein, the Bacillus amyloliquefaciens alpha-amylase of SEQ ID NO: 5 in WO 99/19467, and the Bacillus licheniformis alpha-amylase of SEQ ID NO: 4 in WO 99/19467 and the Bacillus sp. TS-23 alpha-amylase disclosed as SEQ ID NO: 1 in WO 2009/061380 (all sequences are hereby incorporated by reference).
  • the bacterial alpha-amylase may be an enzyme having a degree of identity of at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to any of the sequences shown in SEQ ID NOS: 3, 4 or 5, respectively, in WO 99/19467 and SEQ ID NO: 1 in WO 2009/061380.
  • the alpha-amylase may be an enzyme having a degree of identity of at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to any of the sequences shown in SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 95 herein.
  • the alpha-amylase is derived from Bacillus stearothermophilus.
  • the Bacillus stearothermophilus alpha-amylase may be a mature wild- type or a mature variant thereof.
  • the mature Bacillus stearothermophilus alpha-amylases, or variant thereof, may be naturally truncated during recombinant production.
  • the mature Bacillus stearothermophilus alpha-amylase may be truncated at the C-terminal so it is around 491 amino acids long (compared to SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 95 herein), such as from 480-495 amino acids long.
  • the Bacillus alpha-amylase may also be a variant and/or hybrid. Examples of such a variant can be found in any of WO 96/23873, WO 96/23874, WO 97/41213, WO 99/19467, WO 00/60059, WO 02/10355 and WO2009/061380 (all documents are hereby incorporated by reference). Specific alpha-amylase variants are disclosed in U.S. Patent Nos.
  • BSG alpha-amylase Bacillus stearothermophilus alpha-amylase (often referred to as BSG alpha-amylase) variants having a deletion of one or two amino acids at any of positions R179, G180, 1181 and/or G182, preferably the double deletion disclosed in WO 96/23873 - see, e.g., page 20, lines 1-10 (hereby incorporated by reference), preferably corresponding to deletion of positions 1181 and G182 compared to the amino acid sequence of Bacillus stearothermophilus alpha-amylase set forth in SEQ ID NO: 3 disclosed in WO 99/19467 or SEQ ID NO: 95 herein or the deletion of amino acids R179 and G180 using SEQ I D NO: 3 in WO 99/19467 or SEQ ID NO: 95 herein.
  • BSG alpha-amylase Bacillus stearothermophilus alpha-amylase
  • Bacillus alpha-amylases especially Bacillus stearothermophilus (BSG) alpha-amylases, which have at one or two amino acid deletions corresponding to positions R179, G180, 1181 and G182, preferably which have a double deletion corresponding to R179 and G180, or preferably a deletion of positions 181 and 182 (denoted 1181* + G182*), and optionally further comprises a N193F substitution (denoted 1181* + G182* + N193F) compared to the wild-type BSG alpha- amylase amino acid sequence set forth in SEQ ID NO: 3 disclosed in WO 99/19467 or SEQ ID NO: 95 herein.
  • BSG Bacillus stearothermophilus
  • the bacterial alpha-amylase may also have a substitution in a position corresponding to S239 in the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4 in WO 99/19467, or a S242 variant in the Bacillus stearothermophilus alpha-amylase of SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 95 herein.
  • the variant is a S242A, E or Q variant, preferably a S242Q or A variant, of the Bacillus stearothermophilus alpha-amylase (using SEQ ID NO: 95 herein for numbering).
  • the variant is a position E188 variant, preferably E188P variant of the Bacillus stearothermophilus alpha-amylase (using SEQ ID NO: 95 herein for numbering).
  • Bacillus sp. TS-23 variant disclosed in WO2009/061380 especially variants defined in claim 1 of WO2009/061380 (hereby incorporated by reference).
  • the bacterial alpha-amylase may also be a hybrid bacterial alpha-amylase, e.g. , an alpha-amylase comprising 445 C-terminal amino acid residues of the Bacillus licheniformis alpha-amylase (shown in SEQ ID NO: 4 of WO 99/19467) and the 37 N-terminal amino acid residues of the alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQ ID NO: 5 of WO 99/19467).
  • this hybrid has one or more, especially all, of the following substitutions:
  • variants having one or more of the following mutations (or corresponding mutations in other Bacillus alpha-amylases): H154Y, A181T, N190F, A209V and Q264S and/or the deletion of two residues between positions 176 and 179, preferably the deletion of E178 and G179 (using SEQ ID NO: 5 of WO 99/19467 for position numbering).
  • the bacterial alpha-amylase is the mature part of the chimeric alpha-amylase disclosed in Richardson et al., 2002, The Journal of Biological Chemistry 277(29):. 267501-26507, referred to as BD5088 or a variant thereof.
  • This alpha-amylase is the same as the one shown in SEQ ID NO: 2 in WO 2007134207.
  • the mature enzyme sequence starts after the initial "Met" amino acid in position 1.
  • the alpha-amylase is used in combination with a hemicellulase, preferably xylanase, having a Melting Point (DSC) above 80°C.
  • a hemicellulase preferably xylanase
  • an endoglucanase having a Melting Point (DSC) above 70°C, such as above 75°C, in particular above 80°C may be included.
  • the thermostable alpha-amylase such as a bacterial an alpha-amylase, is preferably derived from Bacillus stearothermophilus or Bacillus sp. TS-23.
  • the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 of at least 10.
  • the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , of at least 15.
  • the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , of at least 20.
  • the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM
  • CaCI 2 of at least 25.
  • the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , of at least 30.
  • the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , of at least 40.
  • the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , of at least 50.
  • the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , of at least 60.
  • the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM
  • the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , between 15-70.
  • the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , between 20-70. In an embodiment the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , between 25-70.
  • the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , between 30-70.
  • the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM
  • the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , between 50-70.
  • the alpha-amylase has a T1 ⁇ 2 (min) at pH 4.5, 85°C, 0.12 mM CaCI 2 , between 60-70.
  • the alpha-amylase is a bacterial alpha-amylase, preferably derived from the genus Bacillus, especially a strain of Bacillus stearothermophilus, in particular the Bacillus stearothermophilus as disclosed in WO 99/19467 as SEQ ID NO: 3 or SEQ ID NO: 95 herein with one or two amino acids deleted at positions R179, G180, 1181 and/or G182, in particular with R179 and G180 deleted, or with 1181 and G182 deleted, with mutations in below list of mutations.
  • the Bacillus stearothermophilus alpha- amylases have double deletion 1181 + G182, and optional substitution N193F, optionally further comprising mutations selected from below list:
  • the bacterial alpha-amylase such as Bacillus alpha-amylase, such as as Bacillus stearomthermphilus alpha-amylase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 95 herein.
  • the bacterial alpha-amylase variant such as Bacillus alpha- amylase variant, such as Bacillus stearomthermphilus alpha-amylase variant has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature part of the polypeptide of SEQ I D NO: 95 herein.
  • Bacillus stearothermophilus alpha- amylase and variants thereof are normally produced naturally in truncated form.
  • the truncation may be so that the Bacillus stearothermophilus alpha-amylase shown in SEQ I D NO: 3 in WO 99/19467 or SEQ I D NO: 95 herein, or variants thereof, are truncated in the C-terminal and are typically around 491 amino acids long, such as from 480- 495 amino acids long.
  • an optional hemicellulase preferably xylanase, having a Melting Point (DSC) above 80°C is present and/or added to liquefaction step i) in combination with an alpha-amylase, such as a bacterial alpha-amylase (described above).
  • DSC Melting Point
  • thermostability of a hemicellulase may be determined as described in the "Materials & Methods' -section under “Determination of Td by Differential Scanning Calorimetry for Endoglucanases and Hemicellulases".
  • the hemicellulase, in particular xylanase, especially GH 10 or GH 1 1 xylanase has a Melting Point (DSC) above 82°C, such as above 84°C, such as above 86°C, such as above 88°C, such as above 88°C, such as above 90°C, such as above 92°C, such as above 94°C, such as above 96°C, such as above 98°C, such as above 100°C, such as between 80°C and 1 10°C, such as between 82°C and 1 10°C, such as between 84°C and 1 10°C.
  • DSC Melting Point
  • the hemicellulase, in particular xylanase, especially GH 10 xylanase has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ I D NO: 96 herein, preferably derived from a strain of the genus Dictyoglomus, such as a strain of Dictyogllomus thermophilum.
  • the hemicellulase, in particular xylanase, especially GH 1 1 xylanase has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 97 herein, preferably derived from a strain of the genus Dictyoglomus, such as a strain of Dictyogllomus thermophilum.
  • hemicellulase in particular xylanase, especially
  • GH10 xylanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 98 herein, preferably derived from a strain of the genus Rasamsonia, such as a strain of Rasomsonia byssochlamydoides.
  • the hemicellulase, in particular xylanase, especially GH10 xylanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 99 herein, preferably derived from a strain of the genus Talaromyces, such as a strain of Talaromyces leycettanus.
  • the hemicellulase, in particular xylanase, especially GH10 xylanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 100 herein, preferably derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus.
  • an optional endoglucanase having a Melting Point
  • thermostability of an endoglucanase may be determined as described in the "Materials & Methods' -section of WO 2017/1 12540 (incorporated herein by reference in its entirety) under the heading "Determination of Td by Differential Scanning Calorimetry for Endoglucanases and Hemicellulases".
  • the endoglucanase has a Melting Point (DSC) above 72°C, such as above 74°C, such as above 76°C, such as above 78°C, such as above 80°C, such as above 82°C, such as above 84°C, such as above 86°C, such as above 88°C, such as between 70°C and 95°C, such as between 76°C and 94°C, such as between 78°C and 93°C, such as between 80°C and 92°C, such as between 82°C and 91 °C, such as between 84°C and 90°C.
  • DSC Melting Point
  • the endogluconase used in a process of the invention or comprised in a composition of the invention is a Glycoside Hydrolase Family 5 endoglucnase or GH5 endoglucanase (see the CAZy database on the "www.cazy.org" webpage.
  • the GH5 endoglucanase is from family EG II , such as the Talaromyces leycettanus endoglucanase shown in SEQ I D NO: 101 herein; Penicillium capsulatum endoglucanase shown in SEQ I D NO: 102 herein, and Trichophaea saccata endoglucanase shown in SEQ I D NO: 103 herein.
  • the endoglucanase is a family GH45 endoglucanase.
  • the GH45 endoglucanase is from family EG V, such as the Sordaria fimicola shown in SEQ I D NO: 104 herein or the Thielavia terrestris endoglucanase shown in SEQ I D NO: 105 herein.
  • the endoglucanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ I D NO: 101 herein.
  • the endoglucanase is derived from a strain of the genus Talaromyces, such as a strain of Talaromyces leycettanus.
  • the endoglucanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ I D NO: 102 herein, preferably derived from a strain of the genus Penicillium, such as a strain of Penicillium capsulatum.
  • the endoglucanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 103 herein, preferably derived from a strain of the genus Trichophaea, such as a strain of Trichophaea saccata.
  • the endoglucanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 104 herein, preferably derived from a strain of the genus Sordaria, such as a strain of Sordaria fimicola.
  • the endoglucanase has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 105 herein, preferably derived from a strain of the genus Thielavia, such as a strain of Thielavia terrestris.
  • the endoglucanase is added in liquefaction step i) at a dose from 1-10,000 ⁇ g EP (Enzymes Protein) /g DS), such as 10-1 ,000 ⁇ g EP/g DS.
  • EP Enzymes Protein
  • an optional carbohydrate-source generating enzyme in particular a glucoamylase, preferably a thermostable glucoamylase, may be present and/or added in liquefaction together with an alpha-amylase and optional hemicellulase, preferably xylanase, having a Melting Point (DSC) above 80°C, and an optional endoglucanase having a Melting Point (DSC) above 70°C, and an optional a pullulanase and/or optional phytase.
  • a glucoamylase preferably a thermostable glucoamylase
  • carbohydrate-source generating enzyme includes any enzymes generating fermentable sugars.
  • a carbohydrate-source generating enzyme is capable of producing a carbohydrate that can be used as an energy-source by the fermenting organism(s) in question, for instance, when used in a process of the invention for producing a fermentation product, such as ethanol.
  • the generated carbohydrates may be converted directly or indirectly to the desired fermentation product, preferably ethanol.
  • a mixture of carbohydrate-source generating enzymes may be used. Specific examples include glucoamylase (being glucose generators), beta-amylase and maltogenic amylase (being maltose generators).
  • the carbohydrate-source generating enzyme is thermostable.
  • the carbohydrate-source generating enzyme in particular thermostable glucoamylase, may be added together with or separately from the alpha-amylase and the thermostable protease.
  • the carbohydrate-source generating enzyme is a thermostable glucoamylase, preferably of fungal origin, preferably a filamentous fungi, such as from a strain of the genus Penicillium, especially a strain of Penicillium oxalicum, in particular the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO 2011/127802 (which is hereby incorporated by reference) and shown in SEQ ID NO: 106 herein.
  • a thermostable glucoamylase preferably of fungal origin, preferably a filamentous fungi, such as from a strain of the genus Penicillium, especially a strain of Penicillium oxalicum, in particular the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO 2011/127802 (which is hereby incorporated by reference) and shown in SEQ ID NO: 106 herein.
  • thermostable glucoamylase has at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the mature polypeptide shown in SEQ ID NO: 2 in WO 201 1/127802 or SEQ ID NOs: 106 herein.
  • the carbohydrate-source generating enzyme in particular thermostable glucoamylase, is the Penicillium oxalicum glucoamylase shown in SEQ ID NO: 106 herein.
  • the carbohydrate-source generating enzyme is a variant of the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO 2011/127802 and shown in SEQ ID NO: 106 herein, having a K79V substitution (referred to as "PE001") (using the mature sequence shown in SEQ ID NO: 14 for numbering).
  • PE001 K79V substitution
  • the K79V glucoamylase variant has reduced sensitivity to protease degradation relative to the parent as disclosed in WO 2013/036526 (which is hereby incorporated by reference).
  • Penicillium oxalicum glucoamylase variants are disclosed in WO 2013/053801 (which is hereby incorporated by reference).
  • these variants have reduced sensitivity to protease degradation.
  • thermostability compared to the parent.
  • the glucoamylase has a K79V substitution (using SEQ ID NO: 106 herein for numbering), corresponding to the PE001 variant, and further comprises at least one of the following substitutions or combination of substitutions: T65A; Q327F; E501V; Y504T; Y504*; T65A + Q327F; T65A + E501V; T65A + Y504T; T65A + Y504*; Q327F + E501V; Q327F + Y504T; Q327F + Y504*; E501V + Y504T; E501V + Y504*; T65A + Q327F + E501V; T65A + Q327F + Y504T; T65A + E501V + Y504T; Q327F + E501V + Y504T; T65A + Q327F + Y504T; T65A + E501V + Y504T; Q
  • Penicillium oxalicum glucoamylase variant has a K79V substitution using SEQ ID NO: 106 herein for numbering (PE001 variant), and further comprises one of the following mutations:
  • the glucoamylase variant such as Penicillium oxalicum glucoamylase variant has at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the mature polypeptide of SEQ ID NO: 106 herein.
  • the carbohydrate-source generating enzyme in particular glycoamylase, may be added in amounts from 0.1- 100 micrograms EP/g DS, such as 0.5-50 micrograms EP/g DS, such as 1-25 micrograms EP/g DS, such as 2-12 micrograms EP/g DS.
  • a pullulanase may be present and/or added during liquefaction step i) together with an alpha-amylase and an optional hemicellulase, preferably xylanase, having a melting point (DSC) above 80°C.
  • an alpha-amylase preferably xylanase, having a melting point (DSC) above 80°C.
  • an optional hemicellulase preferably xylanase, having a melting point (DSC) above 80°C.
  • a protease a carbohydrate-source generating enzyme, preferably a thermostable glucoamylase, may also optionally be present and/or added during liquefaction step i).
  • the pullulanase may be present and/or added during liquefaction step i) and/or saccharification step ii) or simultaneous saccharification and fermentation.
  • Pullulanases (E.C. 3.2.1.41 , pullulan 6-glucano-hydrolase), are debranching enzymes characterized by their ability to hydrolyze the alpha-1 ,6-glycosidic bonds in, for example, amylopectin and pullulan.
  • Contemplated pullulanases include the pullulanases from Bacillus amyloderamificans disclosed in U.S. Patent No. 4,560,651 (hereby incorporated by reference), the pullulanase disclosed as SEQ ID NO: 2 in WO 01/151620 (hereby incorporated by reference), the Bacillus deramificans disclosed as SEQ ID NO: 4 in WO 01/151620 (hereby incorporated by reference), and the pullulanase from Bacillus acidopullulyticus disclosed as SEQ ID NO: 6 in WO 01/151620 (hereby incorporated by reference) and also described in FEMS Mic. Let. (1994) 1 15, 97-106.
  • pullulanases contemplated according to the present invention included the pullulanases from Pyrococcus woesei, specifically from Pyrococcus woesei DSM No. 3773 disclosed in WO 92/02614.
  • the pullulanase is a family GH57 pullulanase.
  • the pullulanase includes an X47 domain as disclosed in WO 2011/087836 (which are hereby incorporated by reference). More specifically the pullulanase may be derived from a strain of the genus Thermococcus, including Thermococcus litoralis and Thermococcus hydrothermalis, such as the Thermococcus hydrothermalis pullulanase shown WO 2011/087836 truncated at the X4 site right after the X47 domain.
  • the pullulanase may also be a hybrid of the Thermococcus litoralis and Thermococcus hydrothermalis pullulanases or a T. hydrothermalis/T. litoralis hybrid enzyme with truncation site X4 disclosed in WO 2011/087836 (which is hereby incorporated by reference).
  • the pullulanase is one comprising an X46 domain disclosed in WO 201 1/076123 (Novozymes).
  • the pullulanase may according to the invention be added in an effective amount which include the preferred amount of about 0.0001-10 mg enzyme protein per gram DS, preferably 0.0001-0.10 mg enzyme protein per gram DS, more preferably 0.0001-0.010 mg enzyme protein per gram DS.
  • Pullulanase activity may be determined as NPUN.
  • An Assay for determination of NPUN is described in the "Materials & Methods"-section below.
  • Suitable commercially available pullulanase products include PROMOZYME 400L, PROMOZYMETM D2 (Novozymes A/S, Denmark), OPTIMAX L-300 (Genencor Int., USA), and AMANO 8 (Amano, Japan).
  • a phytase may be present and/or added in liquefaction in combination with an alpha-amylase and hemicellulase, preferably xylanase, having a melting point (DSC) above 80°C.
  • an alpha-amylase and hemicellulase preferably xylanase, having a melting point (DSC) above 80°C.
  • a phytase used according to the invention may be any enzyme capable of effecting the liberation of inorganic phosphate from phytic acid (myo-inositol hexakisphosphate) or from any salt thereof (phytates).
  • Phytases can be classified according to their specificity in the initial hydrolysis step, viz. according to which phosphate-ester group is hydrolyzed first.
  • the phytase to be used in the invention may have any specificity, e.g., be a 3-phytase (EC 3.1.3.8), a 6-phytase (EC 3.1.3.26) or a 5-phytase (no EC number).
  • the phytase has a temperature optimum above 50°C, such as in the range from 50-90°C.
  • the phytase may be derived from plants or microorganisms, such as bacteria or fungi, e.g., yeast or filamentous fungi.
  • a plant phytase may be from wheat-bran, maize, soy bean or lily pollen. Suitable plant phytases are described in Thomlinson et al, Biochemistry, 1 (1962), 166-171 ; Barrientos et al, Plant. Physiol., 106 (1994), 1489-1495; WO 98/05785; WO 98/20139.
  • a bacterial phytase may be from genus Bacillus, Citrobacter, Hafnia ,
  • Pseudomonas Buttiauxella or Escherichia, specifically the species Bacillus subtilis, Citrobacter braakii, Citrobacter freundii, Hafnia alvei, Buttiauxella gaviniae, Buttiauxella agrestis, Buttiauxella noackies and E. coli.
  • Suitable bacterial phytases are described in Paver and Jagannathan, 1982, Journal of Bacteriology 151 : 1 102-1108; Cosgrove, 1970, Australian Journal of Biological Sciences 23: 1207-1220; Greiner et al, Arch. Biochem.
  • a yeast phytase may be derived from genus Saccharomyces or Schwanniomyces, specifically species Saccharomyces cerevisiae or Schwanniomyces occidentalis.
  • the former enzyme has been described as a Suitable yeast phytases are described in Nayini et al, 1984, Strukturtician und Technologie 17:24-26; Wodzinski et al, Adv. Appl. Microbiol., 42, 263-303; AU-A-24840/95;
  • Phytases from filamentous fungi may be derived from the fungal phylum of Ascomycota (ascomycetes) or the phylum Basidiomycota, e.g., the genus Aspergillus, Thermomyces (also called Humicola), Myceliophthora, Manascus, Penicillium, Peniophora, Agrocybe, Paxillus, or Trametes, specifically the species Aspergillus terreus, Aspergillus niger, Aspergillus niger var. awamori, Aspergillus ficuum, Aspergillus fumigatus, Aspergillus oryzae, T lanuginosus (also known as H.
  • Suitable fungal phytases are described in Yamada et al., 1986, Agric. Biol. Chem. 322: 1275-1282; Piddington et al., 1993, Gene 133:55-62; EP 684,313; EP 0 420 358; EP 0 684 313; WO 1998/28408; WO 1998/28409; JP 7-67635; WO 1998/44125; WO 1997/38096; WO 1998/13480.
  • the phytase is derived from Buttiauxella, such as
  • Buttiauxella gaviniae, Buttiauxella agrestis, or Buttiauxella noackies such as the ones disclosed as SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, respectively, in WO 2008/092901 (hereby incorporated by reference) .
  • the phytase is derived from Citrobacter, such as Citrobacter braakii, such as one disclosed in WO 2006/037328 (hereby incorporated by reference).
  • Modified phytases or phytase variants are obtainable by methods known in the art, in particular by the methods disclosed in EP 897010; EP 897985; WO 99/49022; WO 99/48330, WO 2003/066847, WO 2007/1 12739, WO 2009/129489, and WO 2010/034835.
  • BIO-FEED BIO-FEED
  • PHYTASETM, PHYTASE NOVOTM CT or L all from Novozymes
  • LIQMAX DuPont
  • RONOZYMETM NP RONOZYME® HiPhos
  • RONOZYME® P5000 CT
  • NATUPHOSTM NG 5000 from DSM.
  • a carbohydrate-source generating enzyme preferably a glucoamylase, is present and/or added during saccharification and/or fermentation.
  • the carbohydrate-source generating enzyme is a glucoamylase, of fungal origin, preferably from a stain of Aspergillus, preferably A. niger, A. awamori, or A. oryzae; or a strain of Trichoderma, preferably T reesei; or a strain of Talaromyces, preferably T emersonii,
  • the glucoamylase present and/or added in saccharification and/or fermentation may be derived from any suitable source, e.g., derived from a microorganism or a plant.
  • Preferred glucoamylases are of fungal or bacterial origin, selected from the group consisting of Aspergillus glucoamylases, in particular Aspergillus niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102), or variants thereof, such as those disclosed in WO 92/00381 , WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A.
  • Aspergillus oryzae glucoamylase disclosed in WO 84/02921 , Aspergillus oryzae glucoamylase (Agric. Biol. Chem. (1991), 55 (4), p. 941-949), or variants or fragments thereof.
  • Other Aspergillus glucoamylase variants include variants with enhanced thermal stability: G137A and G139A (Chen et al. (1996), Prot. Eng. 9, 499-505); D257E and D293E/Q (Chen et al. (1995), Prot. Eng. 8, 575-582); N182 (Chen et al. (1994), Biochem. J.
  • glucoamylases include Athelia rolfsii (previously denoted Corticium rolfsii) glucoamylase (see US patent no. 4,727,026 and (Nagasaka et al. (1998) "Purification and properties of the raw-starch-degrading glucoamylases from Corticium rolfsii, Appl Microbiol Biotechnol 50:323-330), Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus (US patent no. Re.
  • the glucoamylase used during saccharification and/or fermentation is the Talaromyces emersonii glucoamylase disclosed in WO 99/28448.
  • Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO 86/01831 ) .
  • Contemplated fungal glucoamylases include Trametes cingulata, Pachykytospora papyracea; and Leucopaxillus giganteus all disclosed in WO 2006/069289; and Peniophora rufomarginata disclosed in WO2007/124285; or a mixture thereof.
  • hybrid glucoamylase are contemplated according to the invention. Examples include the hybrid glucoamylases disclosed in WO 2005/045018. Specific examples include the hybrid glucoamylase disclosed in Table 1 and 4 of Example 1 (which hybrids are hereby incorporated by reference).
  • the glucoamylase is derived from a strain of the genus Pycnoporus, in particular a strain of Pycnoporus as described in WO 2011/066576 (SEQ ID NOs 2, 4 or 6), or from a strain of the genus Gloephyllum, in particular a strain of Gloephyllum as described in WO 201 1/068803 (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16) or a strain of the genus Nigrofomes, in particular a strain of Nigrofomes sp. disclosed in WO 2012/064351 (SEQ ID NO: 2) (all references hereby incorporated by reference).
  • glucoamylases which exhibit a high identity to any of the above- mentioned glucoamylases, i.e., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to any one of the mature parts of the enzyme sequences mentioned above.
  • Glucoamylases may in an embodiment be added to the saccharification and/or fermentation in an amount of 0.0001-20 AGU/g DS, preferably 0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, such as 0.1-2 AGU/g DS.
  • compositions comprising glucoamylase include AMG 200L; AMG 300 L; SANTM SUPER, SANTM EXTRA L, SPIRIZYMETM PLUS, SPIRIZYMETM FUEL, SPIRIZYMETM B4U, SPIRIZYMETM ULTRA, SPIRIZYMETM EXCEL, SPIRIZYMETM ACHIEVE and AMGTM E (from Novozymes A/S); OPTIDEXTM 300, GC480, GC417 (from Genencor Int.); AMIGASETM and AMIGASETM PLUS (from DSM); G-ZYMETM G900, G-ZYMETM and G990 ZR (from Danisco US).
  • the carbohydrate-source generating enzyme present and/or added during saccharification and/or fermentation may also be a maltogenic alpha-amylase.
  • a "maltogenic alpha-amylase” (glucan 1 ,4-alpha-maltohydrolase, E.C. 3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose in the alpha-configuration.
  • a maltogenic amylase from Bacillus stearothermophilus strain NCIB 11837 is commercially available from Novozymes A/S. Maltogenic alpha-amylases are described in US Patent nos. 4,598,048, 4,604,355 and 6, 162,628, which are hereby incorporated by reference.
  • the maltogenic amylase may in a preferred embodiment be added in an amount of 0.05-5 mg total protein/gram DS or 0.05-5 MANU/g DS.
  • the cellulolytic composition used in a process of the invention may be derived from any microorganism.
  • "derived from any microorganism” means that the cellulolytic composition comprises one or more enzymes that were expressed in the microorganism.
  • a cellulolytic composition derived from a strain of Trichoderma reesei means that the cellulolytic composition comprises one or more enzymes that were expressed in Trichoderma reesei.
  • the cellulolytic composition is derived from a strain of Aspergillus, such as a strain of Aspergillus aurantiacus, Aspergillus niger or Aspergillus oryzae.
  • the cellulolytic composition is derived from a strain of Chrysosporium, such as a strain of Chrysosporium lucknowense.
  • the cellulolytic composition is derived from a strain of Humicola, such as a strain of Humicola insolens.
  • the cellulolytic composition is derived from a strain of Penicilium, such as a strain of Penicilium emersonii or Penicilium oxalicum.
  • the cellulolytic composition is derived from a strain of Talaromyces, such as a strain of Talaromyces aurantiacus or Talaromyces emersonii.
  • the cellulolytic composition is derived from a strain of
  • Trichoderma such as a strain of Trichoderma reesei.
  • the cellulolytic composition is derived from a strain of Trichoderma reesei.
  • the cellulolytic composition may comprise one or more of the following polypeptides, including enzymes: GH61 polypeptide having cellulolytic enhancing activity, beta-glucosidase, CBHI and CBHII, or a mixture of two, three, or four thereof .
  • the cellulolytic composition comprising a beta- glucosidase having a Relative ED50 loading value of less than 1.00, preferably less than 0.80, such as preferably less than 0.60, such as between 0.1-0.9, such as between 0.2-0.8, such as 0.30-0.70.
  • the cellulolytic composition may comprise some hemicellulase, such as, e.g., xylanase and/or beta-xylosidase.
  • the hemicellulase may come from the cellulolytic composition producing organism or from other sources, e.g., the hemicellulase may be foreign to the cellulolytic composition producing organism, such as, e.g., Trichoderma reesei.
  • the hemicellulase content in the cellulolytic composition constitutes less than 10 wt.% such as less than 5 wt. % of the cellulolytic composition.
  • the cellulolytic composition comprises a beta-glucosidase.
  • the cellulolytic composition comprises a GH61 polypeptide having cellulolytic enhancing activity and a beta-glucosidase.
  • the cellulolytic composition comprises a beta-glucosidase and a CBH.
  • the cellulolytic composition comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucosidase, and a CBHI.
  • the cellulolytic composition comprises a beta-glucosidase and a CBHI.
  • the cellulolytic composition comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucosidase, a CBHI, and a CBHII.
  • the cellulolytic composition comprises a beta-glucosidase, a CBHI, and a CBHII.
  • the cellulolytic composition may further comprise one or more enzymes selected from the group consisting of a cellulase, a GH61 polypeptide having cellulolytic enhancing activity, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
  • the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
  • endoglucanase is an endoglucanase I.
  • endoglucanase is an endoglucanase II.
  • the cellulolytic composition used according to the invention may in one embodiment comprise one or more beta-glucosidase.
  • the beta-glucosidase may in one embodiment be one derived from a strain of the genus Aspergillus, such as Aspergillus oryzae, such as the one disclosed in WO 2002/095014 or the fusion protein having beta- glucosidase activity disclosed in WO 2008/057637, or Aspergillus fumigatus, such as such as one disclosed in WO 2005/047499 or SEQ ID NO: 107 herein or an Aspergillus fumigatus beta-glucosidase variant, such as one disclosed in WO 2012/044915 or co-pending PCT application PCT/US1 1/054185 (or US provisional application # 61/388,997), such as one with the following substitutions: F100D, S283G, N456E, F512Y.
  • beta-glucosidase is derived from a strain of the genus Penicillium, such as a strain of the Penicillium brasilianum disclosed in WO 2007/019442, or a strain of the genus Trichoderma, such as a strain of Trichoderma reesei.
  • a beta-glucosidase comprising an amino acid sequence having at least 70%, e.g., 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide of SEQ ID NO: 107 herein;
  • a beta-glucosidase encoded by a polynucleotide comprising a nucleotide sequence having at least 70%, e.g. , 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide coding sequence of SEQ ID NO: 5 in WO 2013/148993; and
  • beta-glucosidase encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 5 in WO 2013/148993 or the full-length complement thereof.
  • the beta-glucosidase is a variant comprises a substitution at one or more (several) positions corresponding to positions 100, 283, 456, and 512 of the mature polypeptide of SEQ ID NO: 107 herein, wherein the variant has beta-glucosidase activity.
  • the parent beta-glucosidase of the variant is (a) a polypeptide comprising the mature polypeptide of SEQ ID NO: 107 herein; (b) a polypeptide having at least 80% sequence identity to the mature polypeptide of SEQ ID NO: 107 herein; (c) a polypeptide encoded by a polynucleotide that hybridizes under high or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 5 in WO 2013/148993, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 5 in WO 2013/148993, or (iii) the full-length complementary strand of (i) or (ii); (d) a polypeptide encoded by a polynucleotide having at least 80% identity to the mature polypeptide coding sequence of SEQ ID NO: 5 in WO 2013/148993 or the cDNA sequence thereof; or (e) a fragment
  • the beta-glucosidase variant has at least 80%, e.g., at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%, sequence identity to the amino acid sequence of the parent beta-glucosidase.
  • the variant has at least 80%, e.g., at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 107 herein.
  • the beta-glucosidase is from a strain of Aspergillus, such as a strain of Aspergillus fumigatus, such as Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 107 herein), which comprises one or more substitutions selected from the group consisting of L89M, G91 L, F100D, 1140V, 1186V, S283G, N456E, and F512Y; such as a variant thereof with the following substitutions:
  • the number of substitutions is between 1 and 4, such as 1 , 2, 3, or 4 substitutions.
  • the variant comprises a substitution at a position corresponding to position 100, a substitution at a position corresponding to position 283, a substitution at a position corresponding to position 456, and/or a substitution at a position corresponding to position 512.
  • beta-glucosidase variant comprises the following substitutions: Phel OOAsp, Ser283Gly, Asn456Glu, Phe512Tyr in SEQ ID NO: 107 herein.
  • the beta-glucosidase has a Relative ED50 loading value of less than 1.00, preferably less than 0.80, such as preferably less than 0.60, such as between 0.1-0.9, such as between 0.2-0.8, such as 0.30-0.70.
  • the cellulolytic composition used according to the invention may in one embodiment comprise one or more GH61 polypeptide having cellulolytic enhancing activity.
  • the enzyme composition comprises a GH61 polypeptide having cellulolytic enhancing activity, such as one derived from the genus Thermoascus, such as a strain of Thermoascus aurantiacus, such as the one described in WO 2005/074656 as SEQ ID NO: 2; or one derived from the genus Thielavia, such as a strain of Thielavia terrestris, such as the one described in WO 2005/074647 as SEQ ID NO: 7 and SEQ ID NO: 8; or one derived from a strain of Aspergillus, such as a strain of Aspergillus fumigatus, such as the one described in WO 2010/138754 as SEQ ID NO: 2; or one derived from a strain derived from Penicillium, such as a strain of Penicillium,
  • Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity or homolog thereof is selected from the group consisting of:
  • a GH61 polypeptide having cellulolytic enhancing activity comprising the mature polypeptide of SEQ ID NO: 108 herein;
  • a GH61 polypeptide having cellulolytic enhancing activity comprising an amino acid sequence having at least 70%, e.g., 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide of SEQ ID NO: 108 herein;
  • a GH61 polypeptide having cellulolytic enhancing activity encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 7 in WO 2013/148993 or the full-length complement thereof.
  • the cellulolytic composition used according to the invention may in one embodiment may comprise one or more CBH I (cellobiohydrolase I).
  • the cellulolytic composition comprises a cellobiohydrolase I (CBHI), such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, such as the Cel7A CBHI disclosed in SEQ ID NO: 6 in WO 2011/057140 or SEQ ID NO: 109 herein, or a strain of the genus Trichoderma, such as a strain of Trichoderma reesei.
  • CBHI cellobiohydrolase I
  • a cellobiohydrolase I comprising an amino acid sequence having at least 70%, e.g., 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide of SEQ ID NO: 109 herein;
  • a cellobiohydrolase I encoded by a polynucleotide comprising a nucleotide sequence having at least 70%, e.g., 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%,
  • a cellobiohydrolase I encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 1 in WO 2013/148993 or the full-length complement thereof.
  • the cellulolytic composition used according to the invention may in one embodiment comprise one or more CBH II (cellobiohydrolase II).
  • the cellobiohydrolase II CBHII
  • CBHII cellobiohydrolase II
  • a strain of the genus Aspergillus such as a strain of Aspergillus fumigatus, such as the one in SEQ ID NO: 1 10 herein or a strain of the genus Trichoderma, such as Trichoderma reesei, or a strain of the genus Thielavia, such as a strain of Thielavia terrestris, such as cellobiohydrolase II CEL6A from Thielavia terrestris.
  • a cellobiohydrolase II comprising an amino acid sequence having at least 70%, e.g., 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide of SEQ ID NO: 110 herein;
  • a cellobiohydrolase II encoded by a polynucleotide comprising a nucleotide sequence having at least 70%, e.g., 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the mature polypeptide coding sequence of SEQ ID NO: 3 in WO 2013/148993;
  • a cellobiohydrolase II encoded by a polynucleotide that hybridizes under at least high stringency conditions, e.g., very high stringency conditions, with the mature polypeptide coding sequence of SEQ ID NO: 3 in WO 2013/148993 or the full-length complement thereof.
  • the cellulolytic composition may comprise a number of difference polypeptides, such as enzymes.
  • the cellulolytic composition comprises a Trichoderma reesei cellulolytic composition, further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (WO 2005/074656) and Aspergillus oryzae beta- glucosidase fusion protein (WO 2008/057637).
  • the cellulolytic composition comprises a Trichoderma reesei cellulolytic composition, further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656) and Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499).
  • the cellulolytic composition comprises a Trichoderma reesei cellulolytic composition, further comprising Penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity disclosed in WO 201 1/041397, Aspergillus fumigatus beta- glucosidase (SEQ ID NO: 2 of WO 2005/047499) or a variant thereof with the following substitutions: F100D, S283G, N456E, F512Y.
  • the enzyme composition of the present invention may be in any form suitable for use, such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cellular debris, a semi-purified or purified enzyme composition, or a host cell, e.g., Trichoderma host cell, as a source of the enzymes.
  • the enzyme composition may be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme.
  • Liquid enzyme compositions may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established processes.
  • the cellulolytic composition comprising a beta- glucosidase having a Relative ED50 loading value of less than 1.00, preferably less than 0.80, such as preferably less than 0.60, such as between 0.1-0.9, such as between 0.2-0.8, such as 0.30-0.70.
  • cellulolytic enzyme composition is dosed (i.e. during saccharification in step ii) and/or fermentation in step iii) or SSF) from 0.0001-3 mg EP/g DS, preferably 0.0005-2 mg EP/g DS, preferably 0.001-1 mg/g DS, more preferred from 0.005- 0.5 mg EP/g DS, even more preferred 0.01-0.1 mg EP/g DS.
  • an optional protease such as a thermostable protease
  • a thermostable protease may be present and/or added in liquefaction together with an alpha-amylase, such as a thermostable alpha-amylase, and a hemicellulase, preferably xylanase, having a melting point (DSC) above 80°C, and optionally an endoglucanase, a carbohydrate-source generating enzyme, in particular a glucoamylase, optionally a pullulanase and/or optionally a phytase.
  • an alpha-amylase such as a thermostable alpha-amylase
  • a hemicellulase preferably xylanase, having a melting point (DSC) above 80°C
  • DSC melting point
  • an endoglucanase a carbohydrate-source generating enzyme, in particular a glucoamylase, optional
  • Proteases are classified on the basis of their catalytic mechanism into the following groups: Serine proteases (S), Cysteine proteases (C), Aspartic proteases (A), Metallo proteases (M), and Unknown, or as yet unclassified, proteases (U), see Handbook of Proteolytic Enzymes, A.J.Barrett, N.D.Rawlings, J.F.Woessner (eds), Academic Press (1998), in particular the general introduction part.
  • thermostable protease used according to the invention is a "metallo protease” defined as a protease belonging to EC 3.4.24 (metalloendopeptidases); preferably EC 3.4.24.39 (acid metallo proteinases).
  • protease is a metallo protease or not
  • determination can be carried out for all types of proteases, be it naturally occurring or wild-type proteases; or genetically engineered or synthetic proteases.
  • Protease activity can be measured using any suitable assay, in which a substrate is employed, that includes peptide bonds relevant for the specificity of the protease in question.
  • Assay-pH and assay-temperature are likewise to be adapted to the protease in question. Examples of assay-pH-values are pH 6, 7, 8, 9, 10, or 1 1. Examples of assay- temperatures are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70 or 80°C.
  • protease substrates examples include casein, such as Azurine-Crosslinked Casein (AZCL-casein).
  • AZCL-casein Azurine-Crosslinked Casein
  • Two protease assays are described below in the "Materials & Methods"- section of WO 2017/112540 (incorporated herein by reference), of which the so-called “AZCL-Casein Assay” is the preferred assay.
  • thermostable protease has at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 100% of the protease activity of the JTP196 variant (Example 2 from WO 2017/112540) or Protease Pfu (SEQ ID NO: 11 1 herein) determined by the AZCL-casein assay described in the "Materials & Methods"-section in WO 2017/112540.
  • thermostable protease used in a process or composition of the invention as long as it fulfills the thermostability properties defined below.
  • the protease is of fungal origin.
  • thermostable protease is a variant of a metallo protease as defined above.
  • thermostable protease used in a process or composition of the invention is of fungal origin, such as a fungal metallo protease, such as a fungal metallo protease derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670 (classified as EC 3.4.24.39).
  • thermostable protease is a variant of the mature part of the metallo protease shown in SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 and shown as SEQ ID NO: 112 herein further with mutations selected from below list:
  • thermostable protease is a variant of the mature metallo protease disclosed as the mature part of SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 or SEQ ID NO: 1 12 herein with the following mutations:
  • the protease variant has at least 75% identity preferably at least
  • thermostable protease may also be derived from any bacterium as long as the protease has the thermostability properties defined according to the invention.
  • the thermostable protease is derived from a strain of the bacterium Pyrococcus, such as a strain of Pyrococcus furiosus (pfu protease).
  • protease is one shown as SEQ ID NO: 1 in US patent No. 6,358, 726-B1 (Takara Shuzo Company) and SEQ ID NO: 11 1 herein.
  • thermostable protease is one disclosed in SEQ ID NO: 1 11 herein or a protease having at least 80% identity, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 1 in US patent no. 6,358,726-B1 or SEQ ID NO: 11 1 herein.
  • the Pyroccus furiosus protease can be purchased from Takara Bio, Japan.
  • the Pyrococcus furiosus protease is a thermostable protease according to the invention.
  • the commercial product Pyrococcus furiosus protease (Pfu S) was found (see Example 5 of ) to have a thermostability of 1 10% (80°C/70°C) and 103% (90°C/70°C) at pH 4.5 determined as described in Example 2 of WO 2017/1 12540.
  • the protease has a thermostability of more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, such as more than 105%, such as more than 110%, such as more than 115%, such as more than 120% determined as Relative Activity at 80°C/70°C.
  • protease has a thermostability of between 20 and 50%, such as between 20 and 40%, such as 20 and 30% determined as Relative Activity at 80°C/70°C.
  • the protease has a thermostability between 50 and 115%, such as between 50 and 70%, such as between 50 and 60%, such as between 100 and 120%, such as between 105 and 115% determined as Relative Activity at 80°C/70°C.
  • the protease has a thermostability value of more than 10% determined as Relative Activity at 85°C/70°C determined as described in Example 2 of WO 2017/112540.
  • the protease has a thermostability of more than 10%, such as more than 12%, more than 14%, more than 16%, more than 18%, more than 20%, more than 30%, more than 40%, more that 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 1 10% determined as Relative Activity at 85°C/70°C.
  • the protease has a thermostability of between 10 and 50%, such as between 10 and 30%, such as between 10 and 25% determined as Relative Activity at 85°C/70°C. In an embodiment the protease has more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% determined as Remaining Activity at 80°C; and/or
  • the protease has more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% determined as Remaining Activity at 84°C.
  • the protease may have a themostability for above 90, such as above 100 at 85°C as determined using the Zein-BCA assay as disclosed in Example 3 of WO 2017/112540.
  • the protease has a themostability above 60%, such as above 90%, such as above 100%, such as above 1 10% at 85°C as determined using the Zein-BCA assay.
  • protease has a themostability between 60-120, such as between
  • 70-120% such as between 80-120%, such as between 90-120%, such as between 100- 120%, such as 1 10-120% at 85°C as determined using the Zein-BCA assay.
  • thermostable protease has at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 100% of the activity of the JTP196 protease variant or Protease Pfu determined by the AZCL-casein assay described in the "Materials & Methods"-section of WO 2017/112540.
  • an LPMO polypeptide or enzyme composition comprising at least one LPMO polypeptide for reducing and/or eliminating bacterial contamination in a biofuel fermentation system.
  • an LPMO polypeptide or enzyme composition comprising at least one LPMO polypeptide for reducing and/or eliminating bacterial contamination during yeast propagation.
  • an LPMO polypeptide or enzyme composition comprising at least one LPMO polypeptide for reducing and/or eliminating bacterial contamination in a fermentation medium.
  • an LPMO polypeptide enzyme composition comprising at least one LPMO polypeptide for reducing the levels of lactic acid in a biofuel fermentation system.
  • an LPMO polypeptide or enzyme composition comprising at least one LPMO polypeptide for reducing the levels of lactic acid in a fermentation medium.
  • an LPMO polypeptide or enzyme composition comprising at least one LPMO polypeptide for reducing the levels of lactic acid during yeast propagation.
  • the LPMO polypeptide is selected from the group consisting of
  • Auxiliary Activity 9 (AA9), Auxiliary Activity 10 (AA10), Auxiliary Activity 1 1 (AA11), Auxiliary Activity 13 (AA13), and combinations thereof.
  • the LPMO polypeptide may be a fungal, bacterial, or archeae LPMO polypeptide.
  • the LPMO polypeptide is a fungal AA9 polypeptide.
  • the LPMO polypeptide is a bacterial AA9 polypeptide.
  • the LPMO is an archeae AA9 polypeptide.
  • the LPMO polypeptide is an AA9 selected from the group consisting of:
  • Pe AA9 shown in SEQ ID NO: 2 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto;
  • Tt AA9 shown in SEQ ID NO: 3 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto;
  • the LPMO polypeptide is a fungal AA10 polypeptide. In an embodiment, the LPMO polypeptide is a bacterial AA10 polypeptide. In an embodiment, the LPMO is an archeae AA10 polypeptide.
  • the LPMO polypeptide is a fungal AA11 polypeptide. In an embodiment, the LPMO polypeptide is a bacterial AA11 polypeptide. In an embodiment, the LPMO is an archeae AA1 1 polypeptide.
  • the LPMO polypeptide is a fungal AA13 polypeptide. In an embodiment, the LPMO polypeptide is a bacterial AA13 polypeptide. In an embodiment, the LPMO is an archeae AA13 polypeptide.
  • the LPMO polypeptide is a AA13 polypeptide selected from the group consisting of: i) the Aspergillus terreus AA13 polypeptide of SEQ ID NO: 1 19 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; ii) the Aspergillus lentulus AA13 polypeptide of SEQ ID NO: 120 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; iii) the Aspergillus nidulans polypeptide of SEQ ID NO: 123 or a variant thereof having at least 60%, at least 65%
  • a process for reducing and/or preventing an increase in lactic acid levels in a biofuel fermentation system comprising introducing a LPMO polypeptide or an enzyme composition comprising a lytic polysaccharide monooxygenase (LPMO) polypeptide to a biofuel fermentation system, wherein the fermentation system comprises one or more fermentation vessels, pipes and/or components, and wherein the LPMO polypeptide or enzyme composition comprising the LPMO polypeptide is added at a concentration sufficient to reduce and/or prevent an increase in lactic acid levels in the biofuel fermentation system.
  • LPMO lytic polysaccharide monooxygenase
  • a process for reducing and/or eliminating bacterial contamination in a biofuel fermentation system comprising introducing a LPMO polypeptide or an enzyme composition comprising a lytic polysaccharide monooxygenase (LPMO) to a biofuel fermentation system, wherein the fermentation system comprises one or more fermentation vessels, pipes and/or components, and wherein the LPMO polypeptide or enzyme composition comprising the LPMO polypeptide is added at a concentration sufficient to inhibit growth of contaminating bacterial cells in the biofuel fermentation system.
  • LPMO lytic polysaccharide monooxygenase
  • the LPMO polypeptide is selected from the group consisting of a Auxiliary Activity 9 (AA9) polypeptide, a Auxiliary Activity 10 (AA10) polypeptide, a Auxiliary Activity 11 (AA11) polypeptide, a Auxiliary Activity 13 (AA13) polypeptide, and combinations thereof.
  • the LPMO polypeptide is a AA9 polypeptide selected from the group consisting of:
  • Penicillium emersonii polypeptide of SEQ ID NO: 6 expressed in Trichoderma reesei background or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • Penicillium sp-52627 polypeptide of SEQ ID NO: 142 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto;
  • Penicillium steckii polypeptide of SEQ ID NO: 145 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto;
  • a process for producing a fermentation product from a starch-containing material comprising:
  • At least one LPMO polypeptide or an enzyme composition comprising an LPMO polypeptide is added before or during saccharifying step b) and/or fermenting step c).
  • LPMO polypeptide is selected from the group consisting of a Auxiliary Activity 9 (AA9) polypeptide, a Auxiliary Activity 10 (AA10) polypeptide, a Auxiliary Activity 11 (AA1 1) polypeptide, a Auxiliary Activity 13 (AA13) polypeptide, and combinations thereof.
  • the LPMO polypeptide is a AA13 polypeptide selected from the group consisting of:
  • Penicillium sp-52627 polypeptide of SEQ ID NO: 142 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto;
  • Penicillium steckii polypeptide of SEQ ID NO: 145 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto;
  • the LPMO polypeptide is selected from the group consisting of a Auxiliary Activity 9 (AA9) polypeptide, a Auxiliary Activity 10 (AA10) polypeptide, a Auxiliary Activity 11 (AA11) polypeptide, a Auxiliary Activity 13 (AA13) polypeptide, and combinations thereof.
  • LPMO polypeptide is an AA9 polypeptide selected from the group consisting of:
  • Penicillium emersonii polypeptide of SEQ ID NO: 6 expressed in Trichoderma reesei background or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • LPMO polypeptide is a AA13 polypeptide selected from the group consisting of:
  • the Penicillium polonicum polypeptide of SEQ ID NO: 124 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto;
  • Penicillium sp-52627 polypeptide of SEQ ID NO: 142 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto;
  • Penicillium steckii polypeptide of SEQ ID NO: 145 or a variant thereof having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto;
  • Ta AA9 AA9 polypeptide from Thermoascus aurantiacus having the amino acid sequence of SEQ I D NO: 1.
  • Pe AA9 AA9 polypeptide from Penicillium emersonii having the amino acid sequence of SEQ ID NO: 2.
  • Tt AA9 AA9 polypeptide from Thielavia terrestris having the amino acid sequence of SEQ ID NO: 3.
  • Af AA9 AA9 polypeptide from Aspergillus fumigatus having the amino acid sequence of SEQ ID NO: 4.
  • Tc AA9 AA9 polypeptide from Thermoascus crustaceus having the amino acid sequence of SEQ I D NO: 5.
  • VL-AA9 AA9 polypeptide from Penicillium emersonii expressed in Trichoderma reesei background having the amino acid sequence of SEQ I D NO: 6.
  • At-AA13 AA13 polypeptide from Aspergillus terreus having the amino acid sequence of SEQ I D NO: SEQ I D NO: 1 19.
  • AI-AA13 AA13 polypeptide from Aspergillus lentulus having the amino acid sequence of SEQ I D NO: SEQ I D NO: 120.
  • An-AA13 AA13 polypeptide from Aspergillus nidulans having the amino acid sequence of SEQ I D NO: 123.
  • Pp-AA13 AA13 polypeptide from Penicillium polonicum having the amino acid sequence of SEQ I D NO: 124.
  • PQ-AA13 AA13 polypeptide from Penicillium oxalicum having the amino acid sequence of SEQ I D NO: 125.
  • Mt-AA13 AA13 polypeptide from Mycothermus thermophiles having the amino acid sequence of SEQ I D NO: 127.
  • Alpha-Amylase 369 Bacillus stearothermophilus alpha-amylase with the mutations: I 181 *+G182*+N 193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (SEQ I D NO: 95 herein) truncated to 491 amino acids.
  • Glucoamylase SA Blend comprising Talaromyces emersonii glucoamylase disclosed as SEQ I D NO: 34 in W099/28448, Trametes cingulata glucoamylase disclosed as SEQ I D NO: 2 in WO 06/69289, and Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and starch binding domain (SBD) disclosed in SEQ I D NO: 1 13 herein having the following substitutions G128D+D143N (activity ratio in AGU:AGU: FAU-F is about 20:5: 1).
  • Protease Pfu Protease derived from Pyrococcus furiosus shown in SEQ I D NO: 1 1 1 herein.
  • LPMO's can be used to reduce the levels of bacterial contamination during ethanol fermentation as evidenced by the reduction of levels of a metabolic product of the bacteria present in infected corn mash.
  • LPMO's such as AA9 polypeptides, can reduce the impact of bacterial contamination in corn mash during ethanol fermentation, as evidenced by a reduction in the levels of lactic acid formation in the fermenting mash.
  • Corn mash was prepared in our laboratories under typical liquefaction conditions using a blend of AA369 and Protease Pfu. Infection was found to be undetectable via plating in selective media. Substrate is frozen and thawed before use.
  • Infected corn mash Commercial industrial relevant corn mash with an unknown degree of infection (identified by lactic acid formation and initial cell counts around 10 5 cells/mL) was incubated overnight and used as our source of contamination.
  • Control Clean corn mash plus 1 % infected corn mash was used at 36% dry solids and mixed with urea, to a final concentration of 400 ppm, and commercial glucoamylase GSA, at 0.6 AGU per g dry solids in fermentation. The mix was incubated for 60 minutes at 32°C. Thereafter, yeast was added aiming a pitch of 0.5 g/L in fermentation. Six replicates of fermentations were run for 3 days.
  • Control with commercial antibiotic Clean corn mash plus 1 % infected corn mash was used at 36% dry solids and mixed with urea, to a final concentration of 400 ppm, commercial glucoamylase GSA, at 0.6 AGU per g dry solids in fermentation, and penicillin at 2, 6 or 12 ppm per dry solids. The mix was incubated for 60 minutes at 32°C. Thereafter, yeast was added aiming a pitch of 0.5 g/L in fermentation. Fermentations were run in triplicate for 3 days.
  • Clean corn mash plus 1 % infected corn mash was used at 36% dry solids and mixed with urea, to a final concentration of 400 ppm, commercial glucoamylase GSA, at 0.6 AGU per g dry solids in fermentation, and the AA9 polypeptides listed in the Materials & Methods section above, at 5, 25 or 125 ppm of protein per dry solids.
  • the mix was incubated for 60 minutes at 32°C. Thereafter, yeast was added aiming a pitch of 0.5 g/L in fermentation. Fermentations were run in triplicate for 3 days.
  • FIG. 2 shows lactic acid concentrations after fermentation of corn mash in the presence of various AA9 polypeptides and control (Control, Control with commercial antibiotic).
  • each of the AA9 polypeptides tested reduced the lactic acid formation to a greater extent than in the control (no antibiotic used) and comparable to the positive control in which the largest dosage of penicillin was used.
  • LPMO's such as AA9 polypeptides
  • FIG. 3 shows ethanol concentrations after fermentation of corn mash in the presence of various AA9 polypeptides and control (Control, Control with commercial antibiotic). As shown in FIG.
  • AA9 polypeptides tested improved the ethanol formation to a greater extent than in the control (no antibiotic used) and comparable to the positive control in which the largest dosage of penicillin was used.
  • the LPMO's of the present disclosure can be used during ethanol fermentation to reduce the impact of baseline bacterial contamination as well as infection events caused by lactic acid and acetic acid producting bacteria, aligned with improvements in ethanol production, in biofuel fermentation systems.
  • LPMO's can be used to reduce the impact of bacterial contamination during ethanol fermentation as evidenced by the reduction of levels of a metabolic product of the bacteria present in infected corn mash.
  • LPMO's such as AA13 polypeptides, can reduce lactic acid produced from unwanted bacterial cells presentfrom during ethanol fermentation.
  • Clean liquefied corn mash was prepared in our laboratories under typical liquefaction conditions using a blend of AA369 and Protease Pfu. Infection rate was found to be undetectable via plating in selective media. Substrate is stored frozen and thawed before use.
  • Infected liquefied corn mash Clean corn mash was inoculated with a mixed bacterial population previously isolated from an infected commercial corn mash. The infected mash was incubated with the inoculant for up to 24 hours at approximately 32°C. Final infection rate was found to be greater than 10 8 colony forming units (CFUs) per plating on MRS selective media. Substrate is frozen in 20% glycerol solution and then thawed before use.
  • CFUs colony forming units
  • Infected liquefied mash Per every 100 g of "clean" mash, 1 g of infected mash is added and mixed thoroughly.
  • Positive control Penicillin was used at 25 ppm.
  • Negative control No treatment was added. Yeast, enzymes, and any additional tap water was added at approximately the same time to start fermentation. Fermentations were incubated in a 32°C static water bath for up to 24 hours. All treatments were performed in triplicate.
  • FIG. 4 shows lactic acid concentrations after 24 hours of Low Dry Solids fermentations of corn mash in the presence of various AA13 polypeptides and controls. As shown in FIG. 4, each of the AA13 polypeptides tested reduced the lactic acid formation more than the negative control.
  • polypeptides can reduce the levels of lactic formation in an infected mash during ethanol fermentation, like the AA9 polypeptides in Example 1 above.
  • At-AA13 was selected for additional screening for dose response, and it continued to show reduced lactic acid titers compared to the control after 20 hrs of Low Dry Solids fermentation of corn mash under the above described conditions. These results are shown in FIG. 5.
  • LPMO's can be used to reduce the levels of lactic acid during ethanol fermentation challenged by infection.
  • LPMO's such as AA13 polypeptides, can reduce the levels of lactic acid during ethanol fermentation when challenged by infection.
  • a control mash was prepared in-house with an industry relevant blend of AA369 and Protease Pfu using a Lab-O-Mat incubator for 2 hours at 85°C and 36%DS to simulate typical industry conditions. The mash was then frozen prior to use in SSF. An infected mash was prepared by infecting the control mash with a multi-strain LAB culture grown in MRS. The bacteria in control mash was incubated overnight, and then frozen with 20% glycerol. For this experiment, 1 %weight/weight of the infected mash was mixed into the control mash. This mimics an infection event at a large-scale ethanol facility.
  • SSF SSF
  • All mash was prepared with l OOOppm of urea to aid with yeast fermentation. All treatments were dosed with a baseline commercial glucoamylase GSA, while AA13 candidates were dosed at either 10ug/g-DS or 50ug/g-DS.
  • SSF was performed at 5g scale with 10uL/g rehydrated Ethanol Red yeast at 32°C for up to 24 hours at 20% DS.
  • samples were deactivated with 50uL of 40% sulfuric acid and then centrifuged. The supernatant was filtered through a .2um filter and then measured for soluble carbohydrates and organic acids using an ion-exchange H-column on HPLC.

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EP18797458.9A 2017-10-23 2018-10-19 Verfahren zur verrringerung von milchsäure in einem biokraftstofffermentationssystem Pending EP3701037A1 (de)

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US11795481B2 (en) 2023-10-24
BR112020007814A2 (pt) 2020-10-20
US20210189436A1 (en) 2021-06-24
CA3075907A1 (en) 2019-05-02
US11326187B2 (en) 2022-05-10
US20220267811A1 (en) 2022-08-25
MX2020003981A (es) 2020-08-03
CN111183228A (zh) 2020-05-19
US20240035051A1 (en) 2024-02-01
WO2019083831A1 (en) 2019-05-02

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